The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/β-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.

A bispecific antibody directed to T and B cells (CD3 x CD19 bsAb) was daily infused intravenously in escalating doses from 10 micrograms up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed a t1/2 of 10.5 h with peak levels of 200-300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor alpha was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon gamma. IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1 beta in serum). No gross changes in histology or number of IL-2R+, CD4+ or CD8+ cells were found in the lymph nodes after therapy, but one patient showed activated CD8+ T cells within the tumour nodules. In conclusion, after intravenously administered CD3 x CD19 bsAb only moderate toxicity was found, probably due to CD8+ T cell activation and cytokine release, without CD4+ T cell activation.

Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.

To evaluate the potency by which human T cells are targeted and activated by bispecific monoclonal antibodies (BsAbs) to lyse tumor cells, a clonogenic assay was developed. The efficacy of a CD3 x CD19 BsAb binding to both the CD3 T-cell antigen and the CD19 B-cell antigen was already proven in 51Cr-release assays and in 3-day activation cultures. To achieve more quantitative results, a 14-day clonogenic assay, based on limiting-dilution, was performed for the determination of the initial and residual number of clonogenic units obtained with a CD19+ pre-pre-B acute lymphoblastic leukemia (ALL-B) cell line. Elimination of up to 5 logs of ALL-B cells by freshly isolated peripheral blood mononuclear cells (PBMCs) cultured with BsAb plus interleukin-2 (IL-2) could be detected. The presence of human IgG did not abolish the effect. Repeated addition of each of the two agents was necessary, because a single treatment produced only a 1- to 2-log kill. CD3 monoclonal antibody and IL-2 stimulation (“lymphokine-activated killer cell” conditions) resulted in only a 2-log kill. The number of T cells proved critical in lysis of ALL-B cells, with a 5-log kill using a T-cell:B-cell ratio of 3:1 but with only a 1-log kill using a ratio of 1:1. PBMCs isolated from patients with non-Hodgkin's lymphoma, both in relapse or remission, proved to be as competent as those from healthy donors in removing ALL-B cells. This clonogenic assay shows the importance of repeated administration of CD3 x CD19 BsAb and IL-2 and offers the possibility to compare it with other therapies in B-cell malignancy.