Crystal structure of the human T cell receptor CD3εγ heterodimer complexed to the therapeutic mAb OKT3

Abstract

The CD3␥ heterodimer is essential for expression and function of the T cell receptor. The crystal structure of the human CD3␥ heterodimer is described to 2.1-Å resolution complexed with OKT3, a therapeutic mAb that not only activates and tolerizes mature T cells but also induces regulatory T cells. The mode of CD3␥ dimerization provides a general structural basis for CD3 assembly and maps candidate T cell antigen receptor docking sites, including a duplicated linear region rich in acidic residues that is unique to human CD3. OKT3 binds to an atypically small area of CD3 and has a low affinity for the isolated CD3␥ heterodimer. The structure of the OKT3͞CD3␥ complex has implications for T cell signaling and therapeutic design. T cell recognition is mediated by clonotypically distributed ␣␤ and ␥␦ T cell receptors (TcR) that interact with the peptideloaded molecules of the peptide MHC (pMHC) (1). The antigenspecific chains of the TcR do not possess signaling domains but instead are coupled to the conserved multisubunit signaling apparatus CD3 (2-4). The mechanism by which TcR ligation is directly communicated to the signaling apparatus remains a fundamental question in T cell biology (3, 5). It seems clear that sustained T cell responses involve coreceptor engagement, TcR oligomerization, and a higher order arrangement of TcR-pMHC complexes in the immunological synapse (6-9). However very early TcR signaling occurs in the absence of these events and may involve a ligandinduced conformational change in CD3 (3, 5, 10, 11). The , ␥, ␦ and subunits of the signaling complex associate with each other to form a CD3␥ heterodimer, a CD3␦ heterodimer, and a CD3 homodimer (2). Transfection studies (12), gene knockouts (13), and natural mutations (14) have revealed that the CD3 molecules are important for the proper cell surface expression of the ␣␤ TcR and normal T cell development. The solution structure of the ectodomain fragments of the mouse CD3␥ heterodimer showed that the ␥ subunits are both C2-set Ig domains that interact with each other to form an unusual side-to-side dimer configuration (15). Although the cysteine-rich stalk appears to play an important role in driving CD3 dimerization (15, 16), interaction by means of the extracellular domains of CD3 and CD3␥ is sufficient for assembly of these proteins with TcR␤ (17, 18). Although still controversial, the dominant stoichiometry of the TcR most likely comprises one ␣␤ TcR, one CD3␥ heterodimer, one CD3␦ heterodimer and one CD3 homodimer (2).A number of therapeutic strategies modulate T cell immunity by targeting TcR signaling, particularly the anti-human CD3 mAbs that are widely used clinically in immunosuppressive regimes. The CD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans (19) and is widely used clinically as an immunosuppressive agent in transplantation (20-23), type 1 diabetes (21, 24), and psoriasis (25). Moreover, nonmitogenic anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (26). Anti-CD3 treatment is also reporte...

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

“…In a similar manner, CD3␥ and its glycan are on the ␤ side of the TCR ␣␤ heterodimer. Recent crystal studies of a human CD3 ␥ ectodomain fragment complexed with the Fab of OKT3 and that of human CD3 ␦ complexed with a UCHT1 single-chain Ab fragment identify a similar architecture (25,26).…”

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19+tumor cells

“…Inclusion bodies were isolated, solubilized, refolded and purified via immunoaffinity chromatography, followed by SEC as described. 38 Analysis of purity and stability of AFM11-His The purity, and homogeneity of product samples were assessed by analytical size exclusion chromatography (SE-HPLC) as well as by SDS-PAGE under reducing and non-reducing conditions followed by immunoblotting with His-tag-specific antibodies. Endotoxin content of purified protein was determined by LAL test (Endosafe, Charles River, Wilmington, US).…”

“…Human CD3 antigen was constructed as a CD3ge fusion protein as described. 38 The sequence encoding the fusion construct was synthesized (GeneArt) and subcloned into the bacterial expression vector pET26. Sequence confirmation of both CD19-Fc and CD3ge fusion proteins was performed as described for the TandAb constructs with the exception that for CD3 the reverse primer was 5 0 -GCTAGTTATTGCTCAGCGG-3 0 .…”

Caracterización de la cadena CD3 épsilon en el primate del nuevo mundo Aotus nancymaae

“…la gran variabilidad encontrada en la porción extracelular de la proteína CD3ε de A. nancymaae, los residuos considerados esenciales para la estructura tridimensional están conservados (30)(31)(32).…”

“…De hecho, en los estudios de citometría de flujo en los cuales se han utilizado células mononucleares de sangre periférica de Aotus y anticuerpos monoclonales contra CD3 de humano, no se ha podido encontrar reactividad del anticuerpo (no se presentan los resultados). Los anticuerpos monoclonales específicos contra el CD3 de humano, OKT3 y UCHT1 reconocen un epítopo de conformación producido por aminoácidos ubicados en las interregiones BC, CE y FG de la cadena CD3ε (31,32 En la región transmembrana se encontró en todos los primates comparados un alto porcentaje de identidad en la cadena CD3ε, incluido el residuo ácido (D), esencial para la interacción con residuos básicos que se encuentran en las cadenas α y β del receptor de las células T y que son responsables de la organización del complejo multimérico (35,36).…”