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- PDB-9ip8: Poly-alanine model for HL-type bispecific diabody Ex3 composed of... -

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Basic information

Entry
Database: PDB / ID: 9ip8
TitlePoly-alanine model for HL-type bispecific diabody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilon (closed conformation)
Components
  • Epidermal growth factor receptor
  • HL-type bispecific diabody Ex3
  • T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain
KeywordsANTITUMOR PROTEIN/IMMUNE SYSTEM / bispecific 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 cell-cell adhesion mediated by integrin / positive regulation of T cell anergy / gamma-delta T cell activation / CD4-positive, alpha-beta T cell proliferation / negative thymic T cell selection / positive regulation of CD4-positive, alpha-beta T cell proliferation / alpha-beta T cell receptor complex ...regulation of lymphocyte apoptotic process / gamma-delta T cell receptor complex / T cell anergy / positive regulation of cell-cell adhesion mediated by integrin / positive regulation of T cell anergy / gamma-delta T cell activation / CD4-positive, alpha-beta T cell proliferation / negative thymic T cell selection / positive regulation of CD4-positive, alpha-beta T cell proliferation / alpha-beta T cell receptor complex / positive thymic T cell selection / signal complex assembly / 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 / positive regulation of cell-matrix adhesion / PLCG1 events in ERBB2 signaling / T cell receptor complex / ERBB2-EGFR signaling pathway / smoothened signaling pathway / morphogenesis of an epithelial fold / PTK6 promotes HIF1A stabilization / ERBB2 Activates PTK6 Signaling / digestive tract morphogenesis / Signaling by EGFR / Translocation of ZAP-70 to Immunological synapse / Phosphorylation of CD3 and TCR zeta chains / positive regulation of interleukin-4 production / intracellular vesicle / negative regulation of epidermal growth factor receptor signaling pathway / establishment or maintenance of cell polarity / eyelid development in camera-type eye / dendrite development / cerebral cortex cell migration / protein insertion into membrane / ERBB2 Regulates Cell Motility / protein tyrosine kinase activator activity / alpha-beta T cell activation / Respiratory syncytial virus (RSV) attachment and entry / Signaling by ERBB4 / Generation of second messenger molecules / FCGR activation / PI3K events in ERBB2 signaling / immunological synapse / positive regulation of phosphorylation / Co-inhibition by PD-1 / positive regulation of peptidyl-serine phosphorylation / Estrogen-dependent nuclear events downstream of ESR-membrane signaling / hair follicle development / MAP kinase kinase kinase activity / Role of phospholipids in phagocytosis / GAB1 signalosome / positive regulation of G1/S transition of mitotic cell cycle / embryonic placenta development / salivary gland morphogenesis / T cell receptor binding / T cell costimulation / Signaling by ERBB2 / positive regulation of T cell proliferation / TFAP2 (AP-2) family regulates transcription of growth factors and their receptors / GRB2 events in EGFR signaling / SHC1 events in EGFR signaling / transmembrane receptor protein tyrosine kinase activity / EGFR Transactivation by Gastrin / positive regulation of interleukin-2 production / GRB2 events in ERBB2 signaling / positive regulation of calcium-mediated signaling / ossification / SHC1 events in ERBB2 signaling / FCGR3A-mediated IL10 synthesis / cell surface receptor protein tyrosine kinase signaling pathway / basal plasma membrane / positive regulation of DNA repair / cellular response to epidermal growth factor stimulus / T cell activation / cerebellum development / positive regulation of DNA replication / epithelial cell proliferation / positive regulation of epithelial cell proliferation / Signal transduction by L1 / negative regulation of smoothened signaling pathway / positive regulation of protein localization to plasma membrane / FCGR3A-mediated phagocytosis / NOTCH3 Activation and Transmission of Signal to the Nucleus / cellular response to amino acid stimulus / apoptotic signaling pathway / phosphatidylinositol 3-kinase/protein kinase B signal transduction / cellular response to estradiol stimulus / EGFR downregulation / clathrin-coated endocytic vesicle membrane / calcium-mediated signaling / Signaling by ERBB2 TMD/JMD mutants / Constitutive Signaling by EGFRvIII
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 / Serine-threonine/tyrosine-protein kinase, catalytic domain / Protein tyrosine and serine/threonine kinase / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.91 Å
AuthorsSato, K. / Uehara, S. / Tsugita, A. / Matsui, T. / Asano, R. / Makabe, K. / Yokoyama, T. / Tanaka, Y.
Funding support Japan, 1items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Epidermal growth factor receptor
B: HL-type bispecific diabody Ex3
C: T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain


Theoretical massNumber of molelcules
Total (without water)149,2183
Polymers149,2183
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Epidermal growth factor receptor / Proto-oncogene c-ErbB-1 / Receptor tyrosine-protein kinase erbB-1


Mass: 69496.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EGFR, ERBB, ERBB1, HER1 / Production host: Cricetulus griseus (Chinese hamster)
References: UniProt: P00533, receptor protein-tyrosine kinase
#2: Antibody HL-type bispecific diabody Ex3


Mass: 56781.754 Da / Num. of mol.: 1 / Mutation: Y52W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Brevibacillus choshinensis (bacteria)
#3: Protein T-cell surface glycoprotein CD3 gamma chain,T-cell surface glycoprotein CD3 epsilon chain / T-cell receptor T3 gamma chain / T-cell surface antigen T3/Leu-4 epsilon chain


Mass: 22940.545 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD3G, T3G, CD3E, T3E / Production host: Escherichia coli (E. coli) / References: UniProt: P09693, UniProt: P07766
Has protein modificationN

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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
1HL-type bispecific antibody Ex3 composed of 528 and OKT3 Fvs in ternary complex with sEGFR and CD3gamma-epsilonCOMPLEXall0MULTIPLE SOURCES
2Soluble Epidermal Growth Factor Receptor (sEGFR)COMPLEX#11RECOMBINANT
3HL-type bispecific diabody Ex3COMPLEX#21RECOMBINANT
4CD3 gamma-epsilonCOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
43Homo sapiens (human)9606
52synthetic construct (others)32630
64Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
42Cricetulus griseus (Chinese hamster)10029
53Brevibacillus choshinensis (bacteria)54911
64Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58149 / Symmetry type: POINT

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