Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.

An emerging series of papers has identified new receptor proteins that predict seven-transmembrane pass topologies. We have consolidated this family to 11 human genes and have named the family PAQR, after two of the initially described ligands (progestin and adipoQ receptors). This protein family has ancient evolutionary roots, with identified homologs found in eubacteria. To date, published data indicate that the prokaryotic members of this family appear to encode hemolysin-type proteins, while in eukaryotes, PAQR proteins encode functional receptors with a broad range of apparent ligand specificities. We provide the complete human and mouse complement of this family, suggest a conserved structure/topology with invariant intracellular amino acid residues, and have measured mRNA expression levels for these genes across a range of human tissues.

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 ؋ 10 7 pfu͞mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.Progressive tumor growth in immunocompetent hosts suggests that immune mechanisms are insufficient to detect and eliminate malignant cells. Recent studies indicate that this failure is not always due to the lack of expression of tumor antigens, but rather results from the inability of tumor antigens bound to the major histocompatibility complex (Ag-MHC) to induce adequate T cell proliferation or effector function (1). In this context, it has been shown that the activation of a helper T cell to produce sufficient interleukin 2 (IL-2) to allow autocrinedriven clonal expansion requires costimulatory or accessory signals in addition to T cell receptor (TCR) ligation by Ag-MHC (2-4). The interaction of the CD28 receptor constitutively expressed on T cells with B7-1, a membrane glycoprotein that is induced upon activation on various antigenpresenting cells (5-7), represents one of the best-characterized examples of such a costimulatory signal. Introduction of the B7-1 gene into a variety of murine tumors has been shown to mediate tumor rejection, and in some cases systemic immunity (8), whereas several other poorly immunogenic tumors failed to induce an appropriate immune response (9). B7-1 mediated antitumor activity was largely attributed to its ability to stimulate natural killer (NK) and CD8 ϩ T cells, whereas the requirement of CD4 ϩ cells for tumor rejection is highly dependent on the tu...

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Purpose: This report describes the development and preclinical qualification tests of secondgeneration anti-carcinoembryonic (CEA) designerTcells for use in human trials. Experimental Design: The progenitor first-generation immunoglobulin-T-cell receptor (IgTCR) that transmits Signal 1-only effectively mediated chimeric immune receptor (CIR)^directed cytotoxicity, but expressor T cells succumbed to activation-induced cell death (AICD). The second-generation CIR (termed ''Tandem'' for two signals) was designed to transmit TCR Signal 1 and CD28 Signal 2 to render T cells resistant to AICD and provide prolonged antitumor effect in vivo. Results: A CIR was created that combines portions of CD28,TCR~, and a single chain antibody domain (sFv) specific for CEA into a single molecule (IgCD28TCR). As designed, the genemodified Tandem T cells exhibit the new property of being resistant to AICD, showing instead an accelerated proliferation on tumor contact. Tandem T cells are more potent than first generation in targeting and lysing CEA + tumor. Tandem T cells secrete high levels of interleukin-2 and IFNg on tumor contact that first-generationTcells lacked, but secretion was exhaustible, suggesting a need for interleukin-2 supplementation in therapy even for these second-generation agents. Finally, second-generationTcells were more effective in suppressing tumor in animal models. Conclusion: An advanced generation of anti-CEA designer T cells is described with features that promise a more potent and enduring antitumor immune response in vivo. These preclinical data qualify the human use of this agent that is currently undergoing trial in patients with CEA + cancers.

We have previously demonstrated that intratumoral injection with Ad vectors expressing IL-2 or IL-12 can induce regression in a murine breast cancer model. These IL-2- or IL-12-induced antitumor responses were mainly mediated by Ag-specific T cells. Lymphotactin is a novel lymphocyte chemokine that can cause local accumulation of CD4+, CD8+, and NK cells. We hypothesized that addition of lymphotactin may enhance the antitumor immune responses induced by locally produced IL-2 and IL-12 as we have previously shown. To this end we constructed two double-recombinant adenoviral vectors expressing lymphotactin along with either IL-2 (Ad5 Lym/IL-2) or IL-12 (Ad5 Lym/IL-12). Subcutaneous injection of polyoma middle T (PyMT) or Neu (8142) transgenically derived breast adenocarcinoma cells, in the hind flank of FVB/n mice, results in the formation of tumor nodules in 14-21 days. We show that these constructs elicit potent antitumor responses when administered intratumorally. The antitumor responses are long lasting as determined by rechallenge experiments and hence demonstrate a protective immunity. These observations indicate that by augmenting the antitumor response with adenoviral vectors expressing lymphotactin in combination with IL-2 or IL-12 is a novel way to enhance immunotherapeutic approaches.

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It has been shown that intratumor administration of an adenovirus vector expressing IL-12 produces a potent T cellmediated response that leads to significant tumor regression in a murine breast cancer model. IP-10 and MIG are CXC chemokines that recruit mononuclear cells in vivo. In addition to their chemotactic roles, IP-10 and MIG inhibit angiogenesis. We tested whether the addition of IP-10 or MIG may both enhance the antitumor immune response of IL-12 through T cell recruitment and inhibit tumor growth through angiostasis. Adenovirus vectors expressing IP-10 or MIG and/or IL-12 were administered intratumorally in a