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We searched for cell-surface-associated proteins overexpressed on B cell chronic lymphocytic leukemia (CLL) to use as therapeutic antibody targets. Antibodies binding the immunosuppressive molecule CD200 were identified by cell panning of an antibody phage display library derived from rabbits immunized with primary CLL cells. B cells from 87 CLL patients exhibited 1.6- to 5.4-fold cell-surface up-regulation of CD200 relative to normal B cells. An effect of increased CD200 expression by CLL cells on the immune system was evaluated in mixed lymphocyte reactions. Addition of primary CLL but not normal B cells to macrophages and T cells down-regulated the Th1 response, as seen by a 50-95% reduction in secreted IL-2 and IFN-[gamma] Antibodies to CD200 prevented down-regulation of the Th1 response in most B cell CLL samples evaluated, indicating abrogation of the CD200/CD200R interaction can be sufficient to restore the Th1 response. A disease-progression-associated shift of the immune response from Th1 to Th2 has been observed in numerous cancers. Because this cytokine shift is also believed to promote the induction of regulatory T cells, reverting the immune response to Th1 through direct targeting of the cancer cells may provide therapeutic benefits in CLL by encouraging a cytotoxic T cell response. CD200 | chronic lymphocytic leukemia | immune evasion | immunotherapy

To study regulation in vivo of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of [Beta]-galactosidase under the control of the N-CAM promoter. Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of [Beta]-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals. The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Treatment of the mice with an ampakine, an allosteric modulator of [Alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of [Beta]-galactosidase in vivo as well as in tissue slices in vitro. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.

A large fraction of pediatric pre-B acute lymphoblastoid leukemias (ALL) consistently contain a t(1;19) chromosomal translocation. The t(1;19) translocation results in the production of a chimeric transcription factor containing the N-terminal transactivation domain of E2A fused to the C-terminal DNA-binding homeodomain of Pbx1. Here, we show that the E2A-Pbx1 fusion protein activates the expression of a novel WNT gene, WNT-16. WNT-16 normally is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, but not in bone marrow. In contrast, high levels of WNT-16 transcripts are present in bone marrow and cell lines derived from pre-B ALL patients carrying the E2A-Pbx1 hybrid gene. Inhibition of E2A-Pbx1 expression leads to a significant decrease in WNT-16 mRNA levels, suggesting that WNT-16 is a downstream target of E2A-Pbx1. Three putative WNT receptors, FZ-2, FZ-3, and FZ-5, are expressed in cells of the B lineage, including pre-B ALL cells aberrantly expressing WNT-16. We propose that a WNT-16-mediated autocrine growth mechanism contributes to the development of t(1;19) pre-B ALL.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains., Competing Interests: Competing interest statement: G.D.Y. is a board member, employee, and shareholder of Regeneron Pharmaceuticals, Inc. Other authors are employees and shareholders of Regeneron Pharmaceuticals, Inc.