The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure

The T cell antigen receptor (TCR) consists of a disulfide-linked TCR-alpha/beta heterodimer that is both structurally and functionally associated with a set of four non-covalently linked membrane proteins termed CD3-gamma, -delta, -epsilon, and -zeta. An additional protein described recently, CD3-omega, has been suggested to play a role in assembly of the CD3 complex on the basis of its transient association with the CD3 proteins early during biosynthesis. Association of all the proteins seems to be a prerequisite for intracellular transport, since mutants lacking either the TCR-alpha or -beta protein do not express the CD3 complex on the cell surface. CD3-cDNAs were transfected into COS cells in order to study the protein-protein interactions ruling the assembly of the CD3 macromolecular structure. CD3-delta-epsilon, CD3-gamma-epsilon, and CD3-gamma-delta-epsilon intermediates could be detected. These data indicated that a CD3 core structure could be formed in the absence of the other members of the complex (CD3-zeta, -omega, TCR-alpha, and -beta). Both the individual CD3 chains and the assembled CD3.gamma.delta.epsilon complexes could not be detected on the cellular surface but in an intracellular compartment, probably the endoplasmic reticulum or the cis Golgi. The transfection experiments allowed us to identify the 25-kDa member of the murine CD3 complex as CD3-epsilon m. Furthermore, a 23-kDa glycoprotein seen upon metabolic labeling of human T cells was shown to be an immature form of the CD3-gamma h protein.     

Association of the human CD3-zeta chain with the alpha beta-T cell receptor/CD3 complex. Clues from a T cell variant with a mutated T cell receptor-alpha chain

The TCR for Ag, on the majority of human T cells, is a disulfide-linked heterodimer composed of TCR-alpha and -beta chains noncovalently associated with the monomorphic CD3 complex composed of the CD3-gamma, -delta, -epsilon, and -zeta chains. The interactions involved in the assembly of the various components of this multimeric protein complex are not fully understood. In this report, a variant of the human leukemic T cell line Jurkat that synthesized all of the known components of the TCR/CD3 complex but fails to express the TCR/CD3 complex at the cell surface is further characterized. This variant, J79, has a mutated TCR-alpha chain that does not affect the assembly of the pentameric form (TCR-alpha beta-CD3-gamma delta epsilon) of the TCR/CD3 complex but inhibits the assembly of the CD3-zeta homodimer with the rest of the complex (TCR-alpha beta-CD3-gamma delta epsilon----TCR-alpha beta-CD3-gamma delta epsilon zeta 2). Transfecting a wild-type TCR-alpha gene into J79 reconstituted expression of a complete functionally competent TCR/CD3 complex at the cell surface. The results indicate that the TCR-alpha chain plays a crucial role in the assembly of the CD3-zeta homodimer with the pentameric form of the TCR/CD3 complex.     

Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.     

The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits.

Clonally distributed (clonotypic) antigen receptors on human T lymphocytes (alpha and beta chains) are associated with three invariable T3 polypeptide chains (T3 gamma, delta and epsilon), together forming the T3/T cell receptor complex. Monoclonal antibodies prepared against the two 20-kd T3 polypeptide chains demonstrated that T3-delta and T3-epsilon are distinct polypeptide chains. Only one monoclonal antibody (anti-T3-delta chain) reacted with the T cell surface as judged by indirect immunofluorescence, and by its mitogenicity for quiescent peripheral blood lymphocytes. Immunohistological staining and immunoprecipitation experiments showed that both the T3-delta and T3-epsilon chains are T cell-specific. As seen with the anti-alpha/beta chain reagent WT-31, anti-T3-delta chain monoclonal antibodies stained medullary thymocytes more intensely than cortical thymocytes, whereas the difference between the staining of cortical and medullary thymocytes was generally not apparent with anti-T3-epsilon chain antibodies. Because of this specificity and their ability to react with both the denatured and the native forms of each polypeptide chain, these new monoclonal reagents will be useful tools in studies of the biosynthesis and cell surface expression of the T3/T cell receptor complex during normal and malignant thymic differentiation.     

Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor.

The T-cell antigen receptor (TCR) is a multi-subunit complex consisting of clonotypic heterodimers (TCR-alpha beta or TCR-gamma delta) that are non-covalently linked to at least four invariant chains (CD3-delta, -epsilon, -gamma; and zeta or eta). The ordered process of assembly and the final number of individual chains that comprise the TCR is unclear. In this study, we examined the molecular basis of subunit interactions and the component requirements leading to the formation of a complete TCR. Analysis of transient cotransfections in monkey kidney fibroblasts (COS cells) showed assembly between selective chain pairs. Multiple chain cotransfections demonstrated the formation of stable higher order partial complexes. Assembly of such subcomplexes was facilitated by cooperative interactions between clonotypic and invariant CD3 chains. When zeta was cotransfected with any TCR component, no pairwise interaction was detected. Only when there was coexpression of all of the other TCR chains (TCR-alpha, -beta, CD3- epsilon, -gamma, -delta) did zeta assemble with the TCR complex. Not all chain pairs formed stable heterodimers. For one such pair, lack of assembly is due to the inhibitory effects of negatively charged residues within their transmembrane domains. The combined effects of these interactions probably determine the assembly and the quaternary structure of the TCR complex.     

Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation

The TCR is comprised of two variable chains that confer specificity, called alpha:beta or gamma:delta, physically associated with five different molecules that comprise the complex known as CD3. Antibodies to this complex are very useful, as they react with all T lymphocytes. A rat mAb to mouse CD3 has been prepared. It reacts with 100% of T cells in all mouse strains tested but with no other cell type. It binds to the CD3 epsilon chain. This antibody activates cloned T cell lines and normal T cells, provided suitable accessory cells and signals are present. This antibody detects a determinant similar to but not identical with those detected by two previously reported hamster anti-CD3 epsilon antibodies. This antibody fixes C efficiently, and it is thus useful for depletion of T cells from bulk populations. Activation of T cells by one of the three different anti-CD3 epsilon antibodies was inhibited by the Fab fragment of anti-CD4, similar to the effects of anti-CD4 Fab on two previously reported anti-TCR V region antibodies that bind a CD3 epsilon-associated epitope. This further defines a site involving TCR V regions and CD3 epsilon with which CD4 appears to associate during T cell activation.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

T3-p28 is a protein associated with the delta and epsilon chains of the T cell receptor-T3 antigen complex during biosynthesis

The human T cell receptor-T3 antigen complex is composed of at least five polypeptide chains. In addition to the 45-kDa/50-kDa heterodimer (alpha and beta chains) of the T cell receptor, the complex includes 25-kDa (T3-gamma) and 20-kDa (T3-delta) glycoproteins and a nonglycosylated 20-kDa (T3-epsilon) protein. Here we report that in pulse-chase biosynthetic labeling experiments we detect a new polypeptide chain (T3-p28) which is associated with the T3-delta and T3-epsilon chains during biosynthesis but not on the cell surface. T3-p28, which is not recognized by anti-T3 antibodies, can be chemically distinguished from the previously described T3-gamma chain. The carboxylic ionophore monensin blocks the apparent dissociation of T3-p28 from the T3-delta and T3-epsilon chains. Peripheral blood lymphocytes as well as all T cell leukemic lines tested contain T3-p28, except one HPB-ALL subline. Since the T3-p28 protein is only observed early in biosynthesis of T3-delta and T3-epsilon, it may function in intracellular transport or assembly of the T cell receptor-T3 complex.     

The CD3-gamma delta epsilon transducing module mediates CD38-induced protein-tyrosine kinase and mitogen-activated protein kinase activation in Jurkat T cells.

We have examined the ability of the CD3-gamma delta epsilon and CD3-zeta signaling modules of the T cell receptor (TCR) to couple CD38 to intracellular signaling pathways. The results demonstrated that in TCR+ T cells that express the whole set of CD3 subunits CD38 ligation led to complete tyrosine phosphorylation of both CD3-zeta and CD3-epsilon polypeptide chains. In contrast, in TCR+ cells with a defective CD3-zeta association CD38 engagement caused tyrosine phosphorylation of CD3-epsilon but not of CD3-zeta. Despite these differences, in both cell types CD38 ligation resulted in protein-tyrosine kinase and mitogen-activated protein kinase activation. However, in cells expressing chimerical CD25-zeta or CD25-epsilon receptors or in a TCR-beta- Jurkat T cell line, CD38 ligation did not result in tyrosine phosphorylation of the chimeric receptors, or CD3 subunits, or protein-tyrosine kinase or mitogen-activated protein kinase activation. In summary, these results support a model in which CD38 transduces activating signals inside the cell by means of CD3-epsilon and CD3-zeta tyrosine phosphorylation. Moreover, these data identify the CD3-gamma delta epsilon signaling module as a necessary and sufficient component of the TCR/CD3 complex involved in T cell activation through CD38.     

Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains

Functionally mature human T lymphocytes express a cell-surface receptor for antigen (T cell receptor (TCR)-CD3) composed of at least six polypeptides (TCR-alpha and -beta; T3-gamma, -delta, -epsilon, and -zeta). Immature thymocytes and variants of T cell lines lacking one of the TCR.CD3 polypeptide chains fail to express surface receptor and accumulate the other chains intracellularly. Here we show that the assembly of the TCR.CD3 complex within the endoplasmic reticulum (ER) began with a core of CD3-gamma, -delta, and -epsilon to which TCR-alpha and -beta bound. A recently described intracellular protein, CD3-omega, participated in the assembly since it was found to be associated with the free TCR-alpha or -beta chains or with the CD3 chains. CD3-omega dissociated as TCR.CD3 complexes were formed in the ER. Association of non-disulfide-linked TCR-alpha and -beta chains with CD3 was detected before that of disulfide-bridged TCR-alpha/beta heterodimers. These data suggest that during assembly, the association of TCR-alpha and -beta chains with the CD3 complex precedes the formation of a TCR-alpha/beta dimer. The existence of intermediates consisting of CD3-gamma, -delta, and -epsilon chains and a single TCR-alpha or -beta chain was also confirmed by using a series of variant T cell lines lacking the TCR-beta or -alpha chain, respectively. Once the single TCR-alpha and -beta chains were associated with CD3, disulfide linkages were formed, and a 70-kDa form of the TCR was detected within the ER. This intracellular precursor of the TCR.CD3 complex was subsequently processed into the mature 90-kDa TCR as the TCR.CD3 complex passed through the Golgi apparatus. Assembly of the TCR.CD3 complex is a rather rapid process, whereas export from the ER occurs at a slow rate. After 1 h, 75% of the receptor complex remained within the ER.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

Role of CD2 in regulation of CD3- LGL function.

CD2 is a differentiation marker present on T cells and NK cells. Cytotoxic T lymphocytes (CTL) can be activated by antibodies directed against the CD3/T-cell receptor complex and CD2 structures; however, the role of CD2 in regulation of CD3- large granular lymphocyte (LGL) functions has only recently been studied. Anti-CD2 monoclonal antibodies (mAbs) may be either augmenting or inhibitory and T-cell activation via the CD2 molecule occurs only when mAb binds defined combinations of the CD2 epitopes. Since LGL can be activated by a single stimulus (e.g., IL-2) to proliferate, produce IFN gamma, and increase their cytolytic potential, these functions were chosen to examine the effects of the anti-CD2 mAb and its combinations. Anti-CD2 mAb (D66, GT2, and X11-1) were incubated with LGL for various times in the absence or presence of IL2 and IFN gamma production was monitored. Single anti-CD2 mAb treatment demonstrated minimal augmentation of IFN gamma production. However, combinations of anti-CD2 (9.6) and the other anti-CD2 mAb resulted in a significant, synergistic enhancement of the IFN gamma production. Anti-CD2 mAb treatment appeared to inhibit production generated by optimal doses of IL-2 (1,000 U/ml). The effect of anti-CD2 mAb on IFN gamma production parallel their effects on LGL NK and LAK activity. These data suggested that mAb against the CD2 molecule were important in regulating LGL functions in the absence of a functional CD3 receptor in LGL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an amino acid responsible for the CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis)

The FN18 monoclonal antibody (mAb), directed to CD3 molecules, did not react with the lymphocytes of some cynomolgus monkeys (Macaca fascicularis), because of the polymorphism of the CD3epsilon chain. The epitope recognized by the FN18 mAb was successfully expressed on COS7 cells upon transfection of plasmid DNA coding for the CD3epsilon derived from T cells of a FN18 positive cynomolgus monkey. By construction and expression of plasmid DNA encoding the mutant CD3epsilon, the amino acid residue at position 67 was demonstrated to be involved in the formation of an epitope recognizable by the FN18 mAb.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.     

An enhancer located in a CpG-island 3'' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive.     

Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells

The use of anti-CD3 x antitumor bispecific Abs is an attractive and highly specific approach in cancer therapy. Recombinant Ab technology now provides powerful tools to enhance the potency of such immunotherapeutic constructs. We designed a heterodimeric diabody specific for human CD19 on B cells and CD3epsilon chain of the TCR complex. After production in Escherichia coli and purification, we analyzed its affinity, stability, and pharmacokinetics, and tested its capacity to stimulate T cell proliferation and mediate in vitro lysis of CD19+ tumor cells. The effect of the diabody on tumor growth was investigated in an in vivo model using immunodeficient mice bearing a human B cell lymphoma. The CD3 x CD19 diabody specifically interacted with both CD3- and CD19-positive cells, was able to stimulate T cell proliferation in the presence of tumor cells, and induced the lysis of CD19+ cells in the presence of activated human PBL. The lytic potential of the diabody was enhanced in the presence of an anti-CD28 mAb. In vivo experiments indicated a higher stability and longer blood retention of diabodies compared with single chain Fv fragments. Treatment of immunodeficient mice bearing B lymphoma xenografts with the diabody and preactivated human PBL efficiently inhibited tumor growth. The survival time was further prolonged by including the anti-CD28 mAb. The CD3 x CD19 diabody is a powerful tool that should facilitate the immunotherapy of minimal residual disease in patients with B cell leukemias and malignant lymphomas.