Although bispecific antibodies directed against malignant lymphoma have been shown to be effective in vitro and in vivo, extended clinical trials so far have been hampered by the fact that conventional approaches to produce these antibodies suffer from low yields, ill-defined byproducts, or laborious purification procedures. To overcome this problem, we have generated a small, recombinant, lymphoma-directed, bispecific single-chain (bsc) antibody according to a novel technique recently described. The antibody consists of 2 different single-chain Fv fragments joined by a glycine-serine linker. One specificity is directed against the CD3 antigen of human T cells, and the other antigen-binding site engages the pan–B-cell marker CD19, uniformly expressed on the vast majority of B-cell malignancies. The construct was expressed in Chinese hamster ovary cells and purified by its C-terminal histioline tag. Specific binding to CD19 and CD3 was demonstrated by fluorescence-activated cell sorter analysis. By redirecting unstimulated primary human T cells derived from the peripheral blood against CD19-positive lymphoma cells, the bscCD19 × CD3 antibody showed significant cytotoxic activity at very low concentrations of 10 to 100 pg/mL and at effector to target cell ratios as low as 2:1. Moreover, strong lymphoma-directed cytotoxicity at low antibody concentrations was rapidly induced during 4 hours even in experiments without any T-cell prestimulation. Thus, this particular antibody proves to be much more efficacious than the bispecific antibodies described until now. Therefore, the described bscCD19 × CD3 molecule should be a suitable candidate to prove the therapeutic benefit of bispecific antibodies in the treatment of non-Hodgkin lymphoma.

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V 2 receptor GFP fusion protein (mV 2 R⅐GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC 50 values similar to their K D values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V 2 Rs (hV 2 Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV 2 R.Water homeostasis in mammals is regulated through arginine-vasopressin (AVP), 1 acting through the vasopressin V 2 receptor (V 2 R) expressed in the renal collecting duct (1). In Xlinked nephrogenic diabetes insipidus (NDI), the kidney shows a resistance to the action of AVP, caused by inactivating mutations of the human V 2 R (hV 2 R) gene (2). More than 150 different mutations have been described (for review, see Ref.3), 50% of which are missense mutations resulting in the substitution of a single amino acid. Most of the hV 2 R mutants with a single amino acid exchange are retained within the ER and not transported to the cell surface (for review, see Ref.3). Most likely, the amino acid exchanges result in improper folding of the mutant hV 2 Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established.Recently, it has been found that membrane-permeable antagonists not only inhibit receptor activation, but also promote cell surface expression of misfolded, ER-retained G proteincoupled receptors (GPCRs). This concept represents an intriguing new approach for the therapy of congenital diseases caused by mutations in genes encoding GPCRs. For the ER-retained rhodopsin mutant P23H (a frequent cause of autosomal-dominant retinitis pigmentosa), it has been shown in vitro that the inverse agonist 9-cis-retinal or the non-hydrolyzable inverse agonist 11-cis-7-ring-retinal promoted cell surface expression (5,6). Restoration of cell surface expression by antagonists or inverse agonists has also been...

Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3x10(-9 )towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4x10(6 )for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except approximately 7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

Recently, we have shown that a novel recombinant bispecific single-chain antibody construct (bscCD19 Â CD3), induces highly efficacious lymphoma-directed cytotoxicity mediated by unstimulated peripheral T lymphocytes. Functional analysis of bscCD193CD3 has so far been exclusively performed with human B lymphoma cell lines and T cells from healthy donors. Here we analysed the properties of bscCD193CD3 using primary B cells and autologous T cells from healthy volunteers or patients with B-cell chronic lymphocytic leukaemia (B-CLL). We show that bscCD193CD3 induces T-cell-mediated depletion of nonmalignant B cells in all four cases and depletion of primary lymphoma cells in 22 out of 25 cases. This effect could be observed at low effector-to-target (E:T) ratios and in the majority of cases without additional activation of autologous T cells by IL-2. Even in samples derived from patients heavily pretreated with different chemotherapy regimens, strong cytotoxic effects of bscCD193CD3 could be observed. The addition of bscCD193CD3 to patients' cells resulted in an upregulation of activation-specific cell surface antigens on autologous T cells and elevated levels of CD95 on lymphoma B cells. Although anti-CD95 antibody CH-11 failed to induce apoptosis in lymphoma cells, we provide evidence that B-CLL cell depletion by bscCD3CD3 is mediated at least in part by apoptosis via the caspase pathway.