Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues-which can be chemical conjugation sites-were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

Antibody-enabled small-molecule drug discovery.

Although antibody-based therapeutics have become firmly established as medicines for serious diseases, the value of antibodies as tools in the early stages of small-molecule drug discovery is only beginning to be realized. In particular, antibodies may provide information to reduce risk in small-molecule drug discovery by enabling the validation of targets and by providing insights into the design of small-molecule screening assays. Moreover, antibodies can act as guides in the quest for small molecules that have the ability to modulate protein-protein interactions, which have traditionally only been considered to be tractable targets for biological drugs. The development of small molecules that have similar therapeutic effects to current biologics has the potential to benefit a broader range of patients at earlier stages of disease.

Targeting gastrin-releasing peptide receptors on small cell lung cancer cells with a bispecific molecule that activates polyclonal T lymphocytes.

PURPOSE: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. EXPERIMENTAL DESIGN: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). RESULTS: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNgamma production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule-armed human T cells significantly reduced SCLC tumor burdens in a mouse model. CONCLUSION: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor-positive tumors, such as SCLC, are warranted.

Anti-CD3 antibodies OKT3 and hOKT3gamma1(Ala-Ala) induce proliferation of T cells but impair expansion of alloreactive T cells; aspecifc T cell proliferation induced by anti-CD3 antibodies correlates with impaired expansion of alloreactive T cells.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), has been used for decades to reverse acute transplant rejection. A humanized OKT3 with two alanine replacements on the CH2 domain, hOKT3gamma1(Ala-Ala), was generated to reduce side effects of OKT3. This study reports the effects of OKT3 and hOKT3gamma1(Ala-Ala) on responder T cells in mixed leukocyte cultures (MLC). T cells were purified from peripheral blood mononuclear cells (PBMC) and labeled with CFSE before culture with irradiated allogeneic PBMC in a MLC. Multiparameter flow cytometric analysis of MLC demonstrated logarithmic expansion of T cells with a memory phenotype. Treatment with either OKT3 or OKT3gamma1(Ala-Ala) prevented the logarithmic expansion of these cells. Instead, both OKT3 and hOKT3gamma1(Ala-Ala) induced nonspecific expansion of T cells with both naive and memory phenotype. The proliferating T cells in OKT3 or hOKT3gamma1(Ala-Ala) treated cultures expressed low levels of CD25, and showed a diminished Granzyme B expression compared to that of the untreated MLC, suggesting that these cells may not be fully functional. An array of cytokines in the MLC supernatant was analyzed, and IL-5 and IL-13 were significantly reduced by OKT3 or hOKT3gamma1(Ala-Ala) treatment. These results demonstrated that OKT3 and hOKT3gamma1(Ala-Ala) have similar effects on T cells in MLC. Both antibodies inhibited the logarithmic expansion of allo-reactive T cells, probably through inducing suboptimal proliferation of specific and non-specific T cells. This in vitro study provided one possible mechanism of the therapeutic effects of OKT3 and hOKT3gamma1(Ala-Ala) antibodies.

The production and purification of the human T-cell receptors, the CD3epsilongamma and CD3epsilondelta heterodimers: complex formation and crystallization with OKT3, a therapeutic monoclonal antibody.

Human CD3 is an essential multisubunit complex that plays a fundamental role in T-cell signalling, T-cell development and surface expression of the alphabeta T-cell receptor. The CD3 complex comprises the CD3epsilongamma and CD3epsilondelta heterodimers and the CD3zetazeta homodimer. Here, the expression of the human CD3epsilongamma and CD3epsilondelta heterodimers, both of which were expressed as single-chain polypeptides, is reported. Following refolding, functional heterodimers were immunoaffinity purified from improperly folded heterodimers using OKT3, a therapeutic monoclonal antibody specific for the CD3epsilon chain. Subsequently, the Fab fragment of OKT3 was used to complex individually with the CD3epsilongamma and CD3epsilondelta heterodimers. Crystals of scCD3epsilongamma-FabOKT3 were grown using 15%(w/v) PEG 3350, 200 mM potassium fluoride, 100 mM Tris-HCl pH 8.0. Crystals of scCD3epsilondelta-FabOKT3 were grown using 20%(w/v) PEG 3350, 200 mM potassium formate, 100 mM Tris-HCl pH 8.0, 2%(v/v) MPD. Crystals of both complexes diffract to beyond 3 A resolution. scCD3epsilongamma-FabOKT3 crystals belonged to space group P2(1), with unit-cell parameters a = 67.70, b = 55.77, c = 96.05 A, beta = 100.85 degrees and one complex per asymmetric unit. scCD3epsilondelta-FabOKT3 crystals belong to space group P2(1), with unit-cell parameters a = 101.67, b = 50.36, c = 138.7 A, beta = 108.84 degrees, suggesting two complexes per asymmetric unit.

Crystal structure of the human T cell receptor CD3 epsilon gamma heterodimer complexed to the therapeutic mAb OKT3.

The CD3 epsilon gamma heterodimer is essential for expression and function of the T cell receptor. The crystal structure of the human CD3 epsilon gamma heterodimer is described to 2.1-A 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 epsilon gamma 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 epsilon. OKT3 binds to an atypically small area of CD3 epsilon and has a low affinity for the isolated CD3 epsilon gamma heterodimer. The structure of the OKT3/CD3 epsilon gamma complex has implications for T cell signaling and therapeutic design.

Immunosuppressive properties of anti-CD3 single-chain Fv and diabody.

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains. The linkers used were the natural extensions of the V(H) into the C(H)1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

Activation of human T cells by FcR nonbinding anti-CD3 mAb, hOKT3gamma1(Ala-Ala).

Dimeric Fc receptor (FcR) nonbinding anti-CD3 antibodies have been developed to minimize toxicities associated with classical anti-CD3 monoclonal antibodies (e.g., OKT3). Studies with murine analogs of non-FcR-binding antibodies have shown reduced mitogenicity compared to OKT3. In a trial of an FcR nonbinding humanized anti-CD3 mAb hOKT3gamma1(Ala-Ala) for treatment of patients with type 1 diabetes, we found significant increases in IL-10 and IL-5 in the serum of 63% and 72% of patients, respectively, and TNF-alpha and IL-6 levels that were lower than those previously reported following OKT3 therapy. The activation signal delivered by hOKT3gamma1(Ala-Ala) was associated with calcium signaling and cytokine production by previously activated human cells in vitro. However, the production of IL-10, compared to IFN-gamma on a molar basis, was greater after culture with hOKT3gamma1(Ala-Ala) than with OKT3. Flow cytometric studies confirmed that OKT3 induced IFN-gamma and IL-10 production, but hOKT3gamma1(Ala-Ala) induced only detectable IL-10 production in CD45RO(+) cells. Moreover, in vivo, we found IL-10(+)CD4(+) T cells after drug treatment. These cells were heterogeneous but generally CD45RO(+), CTLA-4(-), and expressed CCR4. A subgroup of these cells expressed TGF-beta. Thus, the non-FcR binding anti-CD3 mAb, hOKT3gamma1(Ala-Ala) delivers an activation signal to T cells that is quantitatively and qualitatively different from OKT3. It leads to the generation of T cells that might inhibit the autoimmune response and may be involved in the beneficial effect on beta cell destruction in Type 1 diabetes.

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites.

A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly(4)-Ser)(3). Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon gamma and tumor necrosis factor alpha, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.

DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression.

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor "mediated uptake is less effected by excessive PEGylation.

Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry.

Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized.

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

In vitro comparison of cytokine release from antithymocyte serum and OKT3. Inhibition with soluble and microencapsulated neutralizing antibodies.

Antilymphocyte antibodies are widely used to prevent and treat rejection after organ transplantation. Induction of cytokine release is implicated in the side effects produced by these antibodies. In this study, cytokine release induced by OKT3 was compared with antithymocyte serum (ATS) using an in vitro whole blood model. The efficacy of the microsphere form of cytokine-neutralizing antibodies to attenuate OKT3 and ATS-induced cytokine release was compared with the soluble form of cytokine-neutralizing antibodies. OKT3-induced significantly higher amounts of tumor necrosis factor-alpha and interleukin 1 beta levels compared with ATS. The microsphere form of tumor necrosis factor-alpha and interleukin 1 beta neutralizing antibodies attenuated OKT3 and ATS-induced cytokine release much more efficiently compared with the soluble form. The results suggest that the severity of the side effects is related to the amount of cytokine release. Microencapsulated cytokine-neutralizing antibodies might be a better therapeutic agent than the soluble form of cytokine-neutralizing antibodies in the treatment of side effects induced by antilymphocyte antibodies.

Cloning and sequencing of the VH and V kappa genes of an anti-CD3 monoclonal antibody, and construction of a mouse/human chimeric antibody.

Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and V kappa) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and V kappa genes to the human heavy and light chain constant region genes (C gamma 1 and C kappa) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.

Selective restimulation of antigen or allergen preactivated T cells using OKT3 F(ab)2 results in the secretion of TH-1 or TH-2-like cytokine patterns.

BACKGROUND: The synthesis of IgE is regulated by cytokines secreted from T-helper cells. The studies on cytokine secretion by peripheral blood mononuclear cells (PBMC) upon stimulation with antigen or allergen are difficult due to low levels of cytokines, especially of interleukin-4 (IL-4). OBJECTIVE: In this study we tried to establish a culture system, which could enable the measurement of the cytokine profiles in specifically activated cultures. METHODS: Three methods to potentiate cytokine secretion were evaluated: PBMC from bee venom or house dust mite (Dermatophagoides pteronyssinus) allergic patients as well as normal subjects were stimulated either with the major bee venom allergen phospholipase A2 (PLA) or with the major D. pteronyssinus allergen (Der p 1) or with the control antigens tetanus toxoid (TT) and purified protein derivate (PPD). After 7 days of culture the cells were restimulated either with plastic bound OKT3 F(ab)2 monoclonal antibodies (MoAbs), with the appropriate antigen + antigen presenting cells or with IL-2. The secretion of cytokines (IL-4, IFN gamma) was measured after restimulation of the cultures (day 8). RESULTS: While OKT3 F(ab)2 was unable to activate resting T cells, it could restimulate preactivated cells. Restimulation with OKT3 F(ab)2 induced higher IL-4 and IFN gamma secretion than restimulation with IL-2 or antigen. TT and PLA stimulated a similar cytokine secretion profile in normal and PLA allergic subjects with substantial levels of both IL-4 and IFN gamma. In contrast, PPD induced virtually only IFN gamma secretion. Der p 1 stimulated mainly IL-4 secretion but also IFN gamma production in some mite allergic patients. CONCLUSION: We have established a cell culture system, which combines antigen specificity with a strong cytokine inducing signal provided by anti-CD3 MoAbs. TH-1 and TH-2 characteristic cytokine patterns can be observed in short-term PBMC cultures already after 8 days of culture.

Sparing of first dose effect of monovalent anti-CD3 antibody used in allograft rejection is associated with diminished release of pro-inflammatory cytokines.

The murine monoclonal antibody OKT3 is the best known of the anti-CD3 antibodies used for the prevention and treatment of renal allograft rejection. Use of this antibody is associated with improved graft outcome but it has a number of adverse effects thought to result from the massive release of pro-inflammatory cytokines. It has been postulated that OKT3 causes cytokine release because of cross-linking of CD3 molecules on the cell surface by bivalent anti-CD3 antibodies, such as OKT3, and the simultaneous binding of the Fc regions of these monoclonal antibodies to Fc receptors on other cells resulting in cell activation. Monovalent antibodies directed against the CD3 antigen should not, in theory, cause cell activation and cytokine release by this postulated mechanism. This study details the use of a monovalent anti-CD3 monoclonal antibody in the treatment of allograft rejection in five renal transplant recipients and documents the degree of TNF, IFN-g and IL6 release generated after antibody injection. Monovalent anti-CD3 monoclonal antibody reversed the rejection episode for which it was used and was well tolerated in all patients. TNF, IFN-g and IL6 measurement showed that little pro-inflammatory cytokine release occurred after this drug. It is likely that the relative lack of side-effects of monovalent anti-CD3 reflects the blunted release of pro-inflammatory cytokines. Monovalent anti-CD3 monoclonal antibody may be a useful addition to the reagents available to treat allograft rejection.

Identification of sites of degradation in a therapeutic monoclonal antibody by peptide mapping.

A peptide mapping procedure was developed to locate regions of a monoclonal antibody, OKT3, that undergo chemical modification as the molecule degrades upon storage. The structures of these peptide degradation products were investigated. Deamidation at specific asparagine residues and oxidation of a cysteine and several methionines were found to be major routes of OKT3 degradation. A unique chain cross-linked degradation product was also observed and characterized. Changing the storage conditions of the antibody affected the relative distribution of degradation products. These results were useful in the development of more stable formulations for OKT3, and the methods can be used in the characterization of other monoclonal antibodies intended for therapeutic use.

Effect of a single amino acid mutation on the activating and immunosuppressive properties of a "humanized" OKT3 monoclonal antibody.

The binding specificity of the murine OKT3 has been transferred into a human antibody framework to reduce its immunogenicity. This "humanized" anti-CD3 mAb (gOKT3-5) was previously shown to retain, in vitro, all the properties of native OKT3, including T cell activation, which has been correlated, in vivo, with the severe side effects observed in transplant recipients after the first administration of the mAb. T cell activation is thought to be triggered by the cross-linking mediated by the antibodies between T cells and Fc receptor-bearing cells. In this study, we introduced a single amino acid mutation from a leucine to a glutamic acid at position 235 in the Fc receptor binding segment of the gOKT3-5 mAb to produce Glu-235 mAb. This mutation generated a 100-fold decrease in the affinity of the antibody for the Fc receptor on U937 cells, without affecting Ag binding. In parallel, we observed a marked reduction in the T cell activation triggered by the mAb (proliferation, cell surface expression of early activation markers including Leu 23 and IL-2R, and release of TNF-alpha, IFN-gamma, and granulocyte macrophage-CSF). In contrast, the mutated mAb retained suppressive properties similar to the gOKT3-5 mAb, as assessed by significant modulation of the T cell receptor complex and suppression of Ag-specific CTL activity. We conclude that this anti-CD3 mAb bearing a single amino acid mutation in its Fc portion retains important immunosuppressive properties, while exhibiting significantly less T cell activation than OKT3 in vitro. This drug might achieve potent immunosuppression while minimizing acute toxicity in vivo and thus be useful in transplantation as well as in autoimmune diseases.

Role of cross-linking in stepwise activation of T cells.

Activation of T cells is a complex process which we have hypothesized involves a series of functional intermediates possessing some, but not all, of the characteristics of fully functional effector cells. We have identified two such functional intermediates, the poised T cell (poTc) and the pre-effector T cell (peTc). poTc do not proliferate or mediate cytolytic activity but are responsive to help in the form of interleukin 2 (IL-2); peTc are proliferating cells which are not cytolytic. Here the role of T-cell receptor/CD3 complex cross-linking in generating these functional intermediates is examined and it is shown that cells can be driven to different stages of functional maturation depending upon the sequence of antibody binding to CD3 and cross-linking. Highly purified T cells can be activated to poTc stage if they are first labelled with the anti-CD3 monoclonal antibody OKT3 and then cross-linked with goat anti-mouse IgG-coated beads. If the OKT3 antibody is first bound to the IgG-coated beads and then added to highly purified T cells, peTc are generated. In both cases the intermediates can be driven to become fully functional effector cells by the addition of IL-2. Finally, by removing the OKT3-bound beads during the activation process, were are able to show that poTc and peTc are sequential intermediates along the same pathway of activation.