Daniel G. Schindler

The T-body approach: potential for cancer immunotherapy

ConclusionsChimeric receptors containing antibody-derived Fv or scFv as their extracellular recognition elements can redirect the specificity of T cells in an MHC-independent manner.Upon encountering their target cells, such T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in both in vitro and in vivo model systems. T cells expressing chimeric receptors are able to discriminate between antigen-expressing and normal cells, with negligible bystander cytotoxicity. Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. Further in vivo studies should be carried out to evaluate and optimize the persistence, homing patterns, and reactivation potential of T-bodies in vivo.Several technical obstacles must be overcome before this approach may be applied clinically. A most urgent problem is the low efficiency of T cell transfection techniques and the particular difficulty of transducing primary T cell populations. While retroviral-mediated gene transfer is more efficient than conventional techniques such as electroporation, the proportion of transfected cells remains low, necessitating an enrichment step. In addition, antibodies with improved discrimination between cell-bound and soluble forms of tumor antigens must be obtained to expand the repertoire of tumor antigens which may be targeted. For each antigen-antibody system, the optimal design of the scFv must be determined.In the future application of this technology, the recognition element used for chimeric TCR is not limited to antibody-derived fragments [27]. Various ligands may be coupled to a T cell-triggering molecule in an attempt to redirect cytotoxic function towards target cells expressing a particular receptor molecule. Although still experimental, we feel that with fine tuning, the T-body approach shows promise as an efficient and broad-spectrum modality for tumor immunotherapy.

Direct T Cell Activation by Chimeric Single Chain Fv-Syk Promotes Syk-Cbl Association and Cbl Phosphorylation

The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signaling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR)·CD3 activation of Src family kinases, we constructed a signaling molecule with an extracellular single chain Fv of an anti-TNP antibody, attached via a transmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, direct aggregation of chimeric Syk with antigen culminates in interleukin-2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-γ (PLC-γ) are phosphorylated in this hybridoma upon TCR·CD3 cross-linking, these two events are uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-γ phosphorylation. These results support a model in which Syk can initiate and directly activate the T cells signaling machinery and position Cbl as a primary tyrosine kinase substrate in this pathway. Furthermore, for efficient PLC-γ phosphorylation to occur in these cells, the combined actions of different tyrosine kinase families may be required.

Differences in the biochemical properties of esterolytic antibodies correlate with structural diversity

A prerequisite to the design and engineering of catalytic antibodies is the knowledge of their structure and in particular which residues are involved in binding and catalysis. We compared the structure and catalytic properties of a series of six monoclonal antibodies which were all raised against a p-nitrophenyl (PNP) phosphonate and which catalyze the hydrolysis of p-nitrophenyl esters. Three of the antibodies (Group I) have similar light and heavy chain variable regions. The other three antibodies have similar VL regions of which two (Group II) have VH regions from the MOPC21 gene family and the remaining one (Group III) a VH from the MC101 gene family making a total of three different groups based on their V region sequences. The structural division into groups is paralleled by the differences in binding constants to hapten analogs, substrate specificity and the susceptibility of the catalytic activity of the antibodies to chemical modification of tryptophan and arginine residues. The relative binding of a transition state analog to the binding of substrate is much higher for the Group I antibodies than for the other groups. Only the Group I antibodies can catalyze the hydrolysis of a carbonate substrate. However all of the antibodies lose catalytic activity upon specific tyrosine modification which highlights the importance of tyrosine in the active site of the antibodies. Thus, antibodies raised against a single hapten can give antibodies with different structures, and correspondingly different specificities and catalytic properties.

Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies

The generation of catalytic antibodies should enable the catalysis of reactions for which no enzymatic or chemical catalyst is currently available. In previous studies, we established a series of catalytic antibodies capable of hydrolysing p-nitrobenzyl (pNB) and p-nitrophenyl (pNP) esters. A group of these catalytic antibodies exhibited high reactivity and substrate specificity, yet each individual antibody demonstrated different kinetic parameters. In order to study the molecular basis for these differences, we have cloned, sequenced and expressed the variable regions of this group of antibodies as functional scFv and Fv in bacteria. The variable region of the heavy chain is derived from a novel germline gene of the J558 family whereas the light chain comes from a germline gene previously found in our catalytic antibodies catalysing the hydrolysis of only nitrophenyl esters, demonstrating that the heavy chain determines the specificity for the nitrobenzyl esters. Several different expression systems were examined for their ability to produce catalytically active antibodies. When expressed as an scFv, both refolded and secreted scFvs exhibited catalytic activity although yields of expressed protein were low. The secreted scFvs had higher specific activity. On the other hand, Fv fragments were expressed in sufficient quantities to allow kinetic analysis. Levels of expression were dependent on the sequence of VL used. Using this expression system, the relative contributions of the individual light and heavy chains to catalysis and binding could be evaluated. Both original VH and VL regions are required for hapten binding, although the VH is more crucial for catalysis. By replacing the CDR3 of the heavy chain with a random sequence, it was shown to be essential for both binding and catalysis. This expression system together with site-directed mutagenesis should enable a more detailed study of the catalytic mechanism of this set of antibodies.

Functional Expression in Mast Cells of Chimeric Receptors with Antibody Specificity

Chimeric genes composed of a single-chain Fv domain (scFv) of an antibody linked with receptor chains normally present in cells of hematopoietic origin were constructed. Such genes could be expressed as functional surface receptors in the RBL-2H3 (rat basophilic leukemia) mast cell line. The chimeric receptors exhibited binding properties of an antibody molecule and triggered degranulation of transfected mast cells on stimulation with antigen. Genetically engineered designer cells (e.g., T-lymphocytes, mast cells, or natural killer cells), equipped with built-in antibody-type recognition, can be now exploited for immunotherapy.