Application of dual affinity retargeting molecules to achieve optimal redirected T-cell killing of B-cell lymphoma.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell-specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell-killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19(-) cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Activity of the rat GluR4 promoter in transfected cortical neurons and glia.

AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptors are assembled from four subunits, GluR1-4. Although GluR4 is widely expressed in brain its abundance is less than GluR1-3. We have isolated approximately 5 kb of the rat GluR4 promoter region and analyzed its capacity to drive expression of a luciferase reporter gene in transfected rat cortical neurons and glia, and C6 glioma cells. Multiple transcriptional start sites were identified in a GC-rich region lacking TATA-boxes between -1090 and -1011 bp from ATG. In transfected mixed cortical cultures, luciferase expression driven by GluR4 promoter segments were found predominantly in TuJ1-positive neurons, indicating neuronal preference of GluR4. The GluR4 promoter fragments were 6-12-fold more active in neurons than glia, compared with a 30-fold neuronal selectivity of GluR2. Deletion of the GluR4 transcriptional initiation region decreased luciferase activity in neurons, but increased activity in C6 cells, suggesting that regulatory elements governing neuronal expression reside in this region. An intron within the 5'-untranslated region and Sp1, IK2 and E-box sites are conserved in the rat, mouse and human GluR4 promoters. The relative activity of GluR4 and GluR2 promoters in transfected cells correlates with their expression in brain, and in both promoters regulatory elements for neuronal expression reside near the initiation sites.

Subunit-dependent modulation of kainate receptors by extracellular protons and polyamines.

Synaptic activity causes significant fluctuations in proton concentrations in the brain. Changes in pH can affect neuronal excitability by acting on ligand-gated channels, including those gated by glutamate. We show here a subunit-dependent regulation of native and recombinant kainate receptors by physiologically relevant proton concentrations. The effect of protons on kainate receptors is voltage-independent and subunit dependent, with GluR5(Q), GluR6(Q), GluR6(R), and GluR6(R)/KA2 receptors being inhibited and GluR6(R)/KA1 receptors being potentiated. Mutation of two acidic residues (E396 and E397) to neutral amino acids significantly reduces the proton sensitivity of the GluR6(Q) receptor, suggesting that these residues influence proton inhibition. The endogenous polyamine spermine potentiated GluR6(R) kainate currents in a pH-dependent manner, producing an acidic shift in the IC(50) for proton inhibition. Spermine potentiation of GluR6(R) is voltage independent, does not affect receptor desensitization, and only slightly shifts the agonist affinity of the receptor. These results suggest that, similar to its action on NMDA receptors, spermine potentiates kainate receptors by relieving proton inhibition of the receptor. Furthermore, they suggest that fluctuations in brain pH during both normal and pathological processes could regulate synaptic transmission and plasticity mediated by kainate receptors.