Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.
CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4 nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.
Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.
2514 Background: CP-870,893 is a fully human IgG2 CD40 agonist antibody currently in early clinical trials. In vitro studies demonstrate its ability to bind human CD40 and enhance dendritic cell costimulatory molecule expression and cytokine production. Methods: In order to assess its potential for cancer therapy, we evaluated the anti-tumor efficacy of CP-870,893 against several CD40pos and CD40neg human tumors in SCIDbeige mice. We specifically addressed the role of tumor CD40 expression, the impact of re-population with human dendritic and T cells on efficacy, and it’s potential to act in synergy with chemotherapeutic agents. Results: We demonstrate that a single i.p. injection of CP-870,893 (T1/2 ∼ 7 days) prevented the growth of several subcutaneous CD40pos tumors including two B cell lymphomas, the breast carcinoma BT-474, and the prostate tumor PC-3 (ED50 = 0.02 mg/kg; Ceff ∼100 ng/mL). Efficacy was demonstrated when CP-870,893 was administered at the time of tumor challenge, but was also observed when treatment was delayed until tumors were well established. Although efficacious against CD40pos tumors, CP-870,893 had no effect on the growth of CD40neg/low tumors unless mice were repopulated at the tumor site with both naïve human dendritic cells and T cells. In these repopulated animals, i.p. administration of CP-870,893 inhibited the growth of a CD40low colon carcinoma and a CD40neg erythroleukemic tumor (ED50 = 0.005 mg/kg). The presence of human T cells and dendritic cells at the tumor site also improved the activity of CP-870,893 against CD40pos tumors, reducing the ED50 and Ceff >10-fold as compared to its effects in the absence of these cells, suggesting a synergy between direct CD40 mediated tumor killing and immune activation. Further, when administered as part of a combination treatment with a sub-optimal dose of cisplatin, improved activity was observed which resulted in tumor regression. Conclusions: These studies demonstrate the potent, broad-spectrum anti-tumor activity of CP-870,893 through both direct and immune mediated effects, its increased efficacy when co-administered with chemotherapeutic agents, and suggest its potential utility as a therapy for human cancer. [Table: see text]
2539 Background: CD40 is expressed on B-cells, monocytes, dendritic cells, other normal tissues and tumors. Previous studies showed that CD40 stimulation enhances antigen presentation, breaks tolerance, bypasses T-cell help, and induces apoptosis in CD40pos tumor cells. We report the in vitro activity and primate pharmacokinetics of a human anti-CD40 agonist antibody, CP-870,893, currently in clinical trials for the treatment of cancer. Methods: CP-870,893 was identified as a CD40 agonist antibody by screening lead molecules generated through the Abgenix Xenomouse® platform. Agonist activity was determined using upregulation of B-cell and monocytes derived dendritic cell surface markers, as well as dendritic cell IL-12 induction. BIAcore and equilibrium binding were utilized to determine affinity, and competition studies with CD40L were conducted on BIAcore. CP-870,893 was administered to cynomolgus monkeys i.v. at various doses, serum antibody levels were evaluated over time in an ELISA assay, and B-cell markers were monitored by FACS. Results: CP-870,893 (IgG2, kappa) binds CD40 with sub-nanomolar affinity, and does not block binding of CD40L. When human whole blood is incubated with CP-870,893, upregulation of key surface molecules involved in antigen presentation (MHC Class II, CD80, CD86, CD23 and ICAM-1) is observed with an EC50 of 5–50 ng/ml. Human monocytes derived dendritic cells, when stimulated with CP-870,893, upregulate activation markers (MHC Class II, CD80 and CD83) with an EC50 of 100–300 ng/ml, and secrete high levels of IL-12p40. In the presence of a second stimulus, such as LPS, human dendritic cells also secreted bioactive IL12-p70 when stimulated with CP-870,893 (EC50 ∼ 150 ng/ml). In addition, a CD40 positive human B-cell tumor line, when stimulated with CP-870,893, becomes susceptible to killing by human CTLs. In cynomolgus monkey studies, the clearance of CP-870,893 decreased with increasing dose. Circulating B-cell numbers decreased, and surface molecules were upregulated on B-cells. Conclusions: These data support the potential utility of CP-870,893 as an immune enhancing agent in cancer immunotherapy, by activating antigen presenting cells, and by enhancing the immunogenicity of CD40 positive tumor cells. [Table: see text]
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