Cancer Gene Therapy (2003) 10, 1 – 13 D 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt Prospects for CD40-directed experimental therapy of human cancer Alex W Tong and Marvin J Stone Cancer Immunology Research Laboratory, Baylor Sammons Cancer Center, Baylor University Medical Center, Dallas, Texas 75246, USA. CD40, a member of the tumor necrosis factor receptor (TNF-R) family, is a surface receptor best known for its capacity to initiate multifaceted activation signals in normal B cells and dendritic cells (DCs). CD40-related treatment approaches have been considered for the experimental therapy of human leukemias, lymphomas, and multiple myeloma, based on findings that CD40 binding by its natural ligand (CD40L), CD154, led to growth modulation of malignant B cells. Recent studies also exploited the selective expression of the CD40 receptor on human epithelial and mesenchymal tumors but not on most normal, nonproliferating epithelial tissues. Ligation of CD40 on human breast, ovarian, cervical, bladder, non small cell lung, and squamous epithelial carcinoma cells was found to produce a direct growth-inhibitory effect through cell cycle blockage and/or apoptotic induction with no overt side effects on their normal counterparts. CD154 treatment also heightened tumor rejection immune responses through DC activation, and by increasing tumor immunogenicity through up-regulation of costimulatory molecule expression and cytokine production of epithelial cancer cells. These immunopotentiating features can produce a ‘‘bystander effect’’ through which the CD40-negative tumor subset is eliminated by activated tumor-reactive cytotoxic T cells. However, the potential risk of systemic inflammation and autoimmune consequences remains a concern for systemic CD154-based experimental therapy. The promise of CD154 as a tumor therapeutic agent to directly modulate tumor cell growth, and indirectly activate antitumor immune response, may depend on selective and/or restricted CD154 expression within the tumor microenvironment. This may be achieved by inoculating cancer vaccines of autologous cancer cells that have been transduced ex vivo with CD154, as documented by recently clinical trials. This review summarizes recent findings on CD154 recombinant protein- and gene therapy–based tumor treatment approaches, and examines our understanding of the multifaceted molecular mechanisms of CD154–CD40 interactions. Cancer Gene Therapy (2003) 10, 1–13 doi:10.1038/sj.cgt.7700527 Keywords: CD40; CD154 (CD40 ligand); gene therapy; immunotherapy; dendritic cells; apoptosis CD40 expression and function in human cells genitors, epithelial and endothelial cells, and all antigen- presenting cells [dendritic cells (DCs), activated monocytes, CD40 was independently identified as a surface marker on activated B lymphocytes, follicular DCs].3 This surface bladder carcinomas and on B cells in 1985.1 Early functional receptor is also found on eosinophils and CD8 + T cells and analysis has established a critical role for this cell surface fibroblasts of synovial membrane and dermal origins.4 receptor for B-cell activation in thymus-dependent humoral Within the thymus, CD40 is expressed on both cortical and responses. The natural ligand for CD40 is a Type II, 39-kDa medullary epithelia and on thymus interdigiting cells.5 By membrane glycoprotein known variously as CD40L, TRAP, comparison, CD154 is expressed transiently on activated and T-BAM. This molecule was defined and isolated in leukocytes, including mature CD4 + T cells, CD8 + T-cell activated T cells in 1992 by CD40–Fc fusion protein and subsets, and g T cells; and mast cells, eosinophils, and IL-2 expression cloning techniques,2 and was subsequently activated NK cells.6 CD154 also has been detected in designated as CD154. purified monocytes, activated B cells, epithelial and vascular CD40 and CD154 expression has since been established in endothelial cells, smooth muscle cells, and DCs, although a wide array of normal hematopoietic and nonhematopoietic the in vivo relevance of CD154 expression in these cell types cell types. CD40 is constitutively expressed on cells with has not been clearly defined.3 The CD40 and CD154 high proliferative potential, including hematopoietic pro- receptor ligand pair is classified correspondingly as members of the tumor necrosis factor receptor (TNF-R) and TNF family of molecules, respectively. The multifaceted nature of CD40 signaling is exemplified Received August 22, 2002. by its diverse physiologic effects in normal B cells. CD40 Address correspondence and reprint requests to: Dr Alex W Tong, + Cancer Immunology Research Laboratory, Baylor Sammons Cancer emerges early on CD34 B-cell precursors in the bone Center, 3500 Gaston Avenue, Dallas, TX 75246, USA. marrow before immunoglobulin gene rearrangement and is E-mail: [email protected] expressed on B cells until their terminal differentiation into CD154 treatment of human cancer AW Tong and MJ Stone 2 plasma cells.7 CD40 ligation led to largely positive growth up-regulated adhesion molecule (E-selectin, VCAM-1/ outcomes for resting B cells, but inhibited the growth and CD54) expression18,26 and triggered proinflammatory cyto- immunoglobulin production of activated B cells.8–12 The kine secretion (IL-1, IL-6, IL-12, interferon/IFN , TNFa) differentiative path of the antigen-activated B cell is by monocytes, fibroblasts, keratinocytes, DCs, smooth ultimately determined by the extent of CD40 activation. muscle cells, and endothelial cells.18 CD154 binding also Abbreviated CD154 exposure skewed the germinal center B modulated the transcription of chemokines and their cell to terminally differentiate into a plasma cell, whereas receptors on macrophages and DCs. CD154 expression on prolonged CD154 exposure generated CD40 + memory B lung and skin mast cells and activated blood basophils may cells.13,14 Patients with CD154 mutation/dysfunction suffer enable these cells to induce IgE secretion by B cells in the from X-linked hyper-IgM immunodeficiency syndrome or presence of IL-4.6 Studies with CD154-blocking reagents common variable hypogammaglobulinemia,15 manifesting and CD40 or CD154 knockouts demonstrated the contribu- as an inability to produce IgG, IgA, and IgE immunoglobulin tion of aberrant CD40 activation to chronic inflammatory isotypes that are needed for an effective, secondary humoral diseases including rheumatoid arthritis,3,27,28 atherosclero- response. Interestingly, these patients suffer from recurrent sis,29,30 neurodegenerative disorders, graft-versus-host dis- pyogenic infections as well as a higher risk of developing ease, and allograft rejection.31,32 Recently, increased lymphomas and gastrointestinal cancers.16,17 CD154–CD40 expression was correlated with elevated Likewise, the initiation and maintenance of cell-mediated cyclooxygenase-2 and nitric oxide synthase at chronic immune responses interaction are critically dependent on the inflammation sites.18,30 CD154 also induced and activated interaction of CD154-expressing, activated T cells with metalloproteinases that, through matrix degradation, may CD40 + DCs.3,18 CD154 produces a prosurvival signal in the be instrumental in tissue destruction observed in rheuma- DC, and up-regulates costimulatory/accessory molecule toid arthritis or atherosclerosis.3 An increased CD154 expression (MHC class II, CD58, CD80/CD86) that en- expression on T cells of patients with multiple sclerosis,33 hances antigen presentation by the CD40 + DC. This inter- systemic lupus erythematosus,34 and myasthenia gravis35 action in turn ‘‘primes’’ CD154 + helper and cytotoxic T cells may contribute to disease pathogenesis by virtue of their by up-regulating their interleukin (IL)-2 receptor expres- proinflammatory features. Conversely, chronic autoimmune sion, and leads to the expansion of both class II– and class inflammatory process and alloimmune responses can be I–dependent tumor-reactive T-cell pools.19 –21 Following abrogated by neutralizing CD40–CD40 ligand interactions, CD154 activation, the antigen-presenting DCs further pro- suggesting that this ligand–receptor pair engenders a central moted cell-mediated immunity by an increased production regulatory role in determining the activation versus tolerance of TNFa, macrophage inflammatory protein (MIP)-1a,IL- outcome of T-dependent immune responses (Table 1). 8, IL-12,22,23 and the recently identified signaling lym- Epithelial CD40 expression is primarily restricted to self- phocytic activation molecule (SLAM) that coordinately renewing stem cells residing in the basal layer, such as basal/ activated neighboring DCs and T cells.24 Another aspect of proliferative layer of nasopharyngeal, tonsillar, and ecto- CD40–CD154 immune regulation is the enhanced cytotoxic cervical epithelium. CD40 binding has produced a uniform capability of IL-2 activated, CD154-expressing NK cells25 cytostatic/growth-inhibitory outcome17 and phenotypic al- after engaging CD40 + target cells. terations that may influence the local inflammatory res- For cells of the reticuloendothelial system, CD40 acti- ponse.36 It has been speculated that activation of CD40, Fas, vation is critical for proinflammatory responses. CD154 or the b1-integrin receptor, all of which are localized on Table 1 In vitro outcome of CD40 receptor binding on normal cells Cell type Outcome Resting B cells Isotype switching, maturation to memory B cell or plasma cell; cytokine secretion, CD23, VLA-4, CD80/86 up-regulation Activated B cells