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 Growth inhibition and increased apoptosis, increased Fas/Apo-1 expression Monocyte/macrophages Decreased apoptosis; up-regulated expression of CD54, CD86, CD40, MHC Class II; increased cytokine secretion (IL-1b, IL-6, IL-8, IL-12, TNFa,IFNg) and nitric oxide production Monocytic dendritic cells Increased survival; cytokine (IL-1, IL-6, IL-8, IL-10, IL-12, TNFa,MIP-1a) and enzyme (MMP) secretion; up-regulation of costimulatory molecules (CD54/ICAM-1, CD58/LFA-3, CD80/B7-1, CD86/B7-2) Follicular dendritic cells Growth stimulation T cells Proliferation; IL-2 receptor up-regulation Endothelial cells Activation, up-regulation of CD54/ICAM-1, CD106/ VCAM-1, CD62E/E-selectin, enhancing leukocyte adherence Basal epithelium Cytostatic/growth inhibition Thymic epithelial cells GM-CSF secretion, growth inhibition Kidney epithelial cells RANTES secretion Keratinocytes Growth inhibition; IL-6, IL-8, TNFa secretion; CD54 up-regulation Fibroblasts IL-6 production, growth stimulation VLA: very late antigen; TNF: tumor necrosis factor; IFN: interferon; MIP: macrophage inflammatory protein; MMP: matrix metalloproteinase; ICAM: intracellular adhesion molecule; LFA: leukocyte functional antigen; VCAM: vascular cell adhesion molecule; GM-CSF: granulocyte– macrophage colony-stimulating factor; RANTES: regulated on activation, normal T cell expressed and secreted.

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 3 basil epithelium,37 may direct the self-renewing epithelial In contrast, CD40 cross-linking induced cell cycle arrest in cells towards a prodifferentiative path. As CD154 was rarely murine (BCL1) and human (Daudi) B lymphoma cells, detected in normal and malignant epithelial cells, CD40 which was critical for the induction and maintenance of regulation probably originated from CD154 + , infiltrating T tumor dormancy.47 For posttransplant, Epstein-Barr virus cells, particularly g T cells that congregate preferentially at (EBV)–positive high-grade aggressive lymphomas, CD40 the epithelium during inflammation. Alternatively, a CD40 binding led to growth inhibition through the induction of ligand analog that is unique to epithelial sites may be apoptosis.48 Growth arrest, up-regulation of Fas, and expressed.17 increased apoptosis16,47 were similarly observed in vitro CD40 expression on malignant cells generally mirrors following CD40 binding of non-HIV, high-grade malig- their normal, proliferating counterparts, and has been nancies such as Burkitt’s lymphoma.44 We and others detected on B-cell malignancies, melanomas, and a variety showed that human multiple myeloma cells also expressed of carcinoma cell types.17,38,39 Based on the growth- and CD40,49,50 and underwent growth arrest upon treatment with immune-regulatory features of CD154–CD40 interaction in soluble, recombinant CD154.10,17,50,51 A bimodal growth- normal cells, agonistic anti-CD40 monoclonal antibodies regulatory process for myeloma cells has been suggested (mAbs), recombinant CD154, or CD154 gene transfer by Teoh et al,52 where CD40 ligation activated myeloma agents have been considered to promote corresponding cells in the initial phase followed by induction of apoptosis, direct and indirect tumor growth-inhibitory outcomes. These with a correspondingly distinct compliment of gene sets that findings are summarized in the following sections, which is related to cell cycle progression, or apoptosis that is also examine the findings of recent Phase I/II cancer significantly enhanced by Apo-1/Fas (CD95) ligation.53 experimental therapy trials with recombinant CD154 and CD40 ligation appears to play a growth-inhibitory/ CD154 gene transfer, and our current understanding of prodifferentiative role for normal as well as malignant molecular pathways that mediate the diverse cellular epithelial cells. In the oral basal epithelium, >80% of total outcomes following CD40 ligation. cells coexpressed CD40 and CD154. A loss of CD154 expression and maintained expression of CD40 may con- tribute to epithelial tumorigenesis, as observed recently Direct growth inhibition of cancers in squamous cell carcinoma.54 – 56 Consistent with this hy- pothesis, patients with X-linked hyper-IgM syndrome also Much like the diverse regulatory effects of CD40 on normal suffered from an increased incidence of developing carci- cells, CD154 produced a wide range of growth-regulatory nomas of the liver, pancreas, and biliary tree.16,17 The loss of effects on CD40-expressing tumor cells (Table 2). In CD154-dependent epithelial cell growth regulation, or un- Hodgkin’s disease and low-grade B-cell malignancies such controlled chronic infection and inflammation, has been as follicular lymphomas,40,41 hairy cell leukemia,42 and B suggested by Young et al17 to contribute to increased tumor chronic lymphocytic leukemias (B-CLL43 ), CD40 activa- susceptibility. tion contributed to tumor survival and resistance to chemo- CD40 expression has been documented in nasopharyn- therapy.9,44 CD40 binding triggered the growth of human geal, colorectal, bladder, ovarian, liver, and breast carcino- immunodeficiency virus (HIV)–related lymphomas, possi- mas, whereas CD154 was not endogenously expressed, or bly through increased vascularization and activation of expressed at very low levels. Strong expression of CD40 in nuclear factor kappa of B cells (NF-kB), which in turn the tumor vasculature of renal57 and breast58 carcinomas stimulated HIV replication.45 Recent preliminary findings of suggests a potential role of this receptor in tumor angio- Aldinucci et al46 identified CD40 expression in 84% of genesis. In a concurrent evaluation of CD40 and CD154 patients with acute myelomonocytic leukemia. Exposure to expression, we found that CD40 was expressed on all soluble CD40 ligand led to dose-dependent proliferation and primary breast tumor histologic subtypes, including infiltrat- clonogenic colony formation of the leukemia blasts in vitro. ing ductal and lobular carcinomas, and carcinomas in situ.58

Table 2 Varying direct growth-modulatory effects of CD40 ligation on human tumors

Histology type Growth outcome References

Carcinomas (bladder, ovarian, # proliferation, " apoptosis [17,38,54,55,58,59,62,68,69,70,95] cervical, squamous cell, lung) Melanoma " apoptosis [60] Acute myelomonocytic leukemia " proliferation [46] Low-grade B cell malignancies " proliferation, # apoptosis, " apoptosis [16] in NHL but not CLL High-grade B cell leukemias and lymphomas # proliferation, " apoptosis [12,16,40–43,47] Burkitt’s lymphoma " or # apoptosis [16] HIV-related lymphoma " proliferation [16,45] B-cell acute lymphoblastic leukemia " cell cycle activity [16] Multiple myeloma " or # proliferation, " apoptosis [10,49–53] Hodgkin’s disease " clonogenicity, resistance to chemotherapy [9,44] (") Increased; (#) decreased. NHL: non-Hodgkin’s lymphoma; CLL: chronic lymphocytic leukemia.

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 4 Although focal areas of the tumor populations also expressed demonstrated a significant antitumor effect that was additive CD154, this molecule was detected primarily within the with cisplatin and unaffected by cotreatment with antiasialo cancer cell cytoplasm with rare membrane staining, sug- GM-1 that blocked NK activity.68 These findings confirmed gesting that expression of the membrane-bound CD154 may the direct growth-inhibitory effect of rhuCD40L on be uncommon, or occurs transiently (and hence escapes epithelial ovarian tumors. detection). Restricted endogenous CD154 expression may Like the normal epithelium,17 CD40 expression on breast limit its impact towards regulating disease progression.58 carcinoma cells can be up-regulated by IFNg treatment, Further, CD154 was rarely expressed among tumor- which was shown to synergize with an agonistic anti-CD40 infiltrating lymphocytes (TILs) in the majority of breast mAb in inhibiting the growth of SCID-hu breast cancer cell cancer cases tested, suggesting that TILs in established line xenografts.62 Wingett et al69 showed that IFNg pre- breast cancers may lack the ability to down-regulate tumor activation was required for a significant, CD40-dependent cell growth through CD40L–CD40 interaction.58 antitumor response in vivo. By contrast, we58 and others62 A direct growth-inhibitory effect through CD40 binding observed that IFNg production was not critical for CD154- of epithelial cancers was first demonstrated by Hess and induced tumor growth inhibition, although an enhancing Engelmann,38 who showed that CD154 transgene expression effect by this and other cytokines cannot be excluded.62,70 in HeLa cervical carcinoma cells dramatically reduced The findings described above indicate that CD154 can growth rate in vitro. This CD40-mediated growth-inhibitory regulate cell growth in positive and negative directions: effect did not appear to be mediated by apoptosis in studies CD40 signaling rescues normal B cells, monocytes, DCs, of bladder, ovarian, and skin carcinomas, and was additive and fibroblasts from cell death, but promotes apoptosis in with the proapoptotic activities of cytotoxic cisplatin and transformed cells of mesenchymal and epithelial origin as other biologic response modifiers including TNFa, Fas well as neuronal cells and hepatocytes. The triggering of ligation, and ceramide.54 Recently, human non small cell other receptors (p55 and p70 TNF-receptors, CD30, CD19) lung carcinomas were also found to express CD40. CD154 may similarly activate normal cells while inhibiting the treatment caused dose-dependent, reversible cell cycle S- growth of transformed cells. This phenomenon has been phase blockage in the tumor subset that expressed a high termed ‘‘activation-induced cell death’’ (AICD)71 in the level of CD4059 — a mechanism that was similar to its cell context of immune cell interactions, where the circumstance cycle blockade of cisplatin-resistant ovarian cancer cells.17 of cell activation versus inhibition appeared to depend on the For breast and squamous carcinoma lines and SV40- coordinate activation of other surface molecules (such as the transformed keratinocytes, apoptotic induction appeared to antigen receptor) and physiologic state of the receptor- play a critical role in CD40-dependent growth-inhibitory expressing cell (proliferation versus quiescence, intracellular effect (Table 254). CD154 also inhibited melanoma cell pro- reducing/oxidating state balance).72 – 75 As members of the liferation in vitro through apoptotic induction,60 enhanced TNF-R superfamily, CD40, CD30, and the TNF-Rs share tumor-specific lymphocyte-mediated tumor cytotoxicity, the common feature of lacking a cytoplasmic domain with and stimulated the production of immune-activating cyto- intrinsic kinase or other well-defined signal transduction kines by melanoma cells that included IL-6, IL-8, and capacity. Rather, CD154 binding induced CD40 homotrime- TNFa. rization without altering its conformation.65,76 – 78 This event Studies with human B lymphoma xenografts in severe induces oligomerization of members of the TRAF (TNF-R combined immunodeficiency (SCID) mice validated the in associated factor) family of signal transducers that bind to vivo tumor-inhibitory properties of CD154 in the absence of two conserved, complementary CD40 cytoplasmic domains T- and B-cell activation.16,61 We and others observed a (TRAF domains78,79 ). Of the six TRAF members identified similar antitumorigenic effect on CD40 + human breast to date (TRAF1–6), TRAF3 is most prominently associated cancer cell line xenografts in SCID mice,58,62 for which with CD40 signaling, and competes with TRAF1 and different constructs of soluble CD154 produced a uniform TRAF2 for a TRAF domain distal to the plasma mem- growth-inhibitory effect. Agents that were evaluated in- brane.79,80 TRAF5 may also associate at this domain through cluded gp39, a CD40L–CD8 recombinant molecule,63 the interaction with the RING finger domain of TRAF2 and trimeric human CD40 ligand/leucine zipper fusion protein TRAF3.81 A fifth member, TRAF6, associates with a second huCD40LT,64 and the recombinant rhsCD40L of similar CD40 TRAF domain proximal to the plasma membrane and construct without a leucine zipper.65 Cotreatment with the may serve to modulate TRAF1, TRAF2, TRAF3, or TRAF5 CD154-blocking antibody LL48 abrogated this antitumor binding outcome.77,79 The homo- and heterooligomerization effect in vitro and in vivo, confirming CD40 dependence of of individual TRAFs in turn activate unique as well as this phenomenon. Conversely, CD154 was ineffective in overlapping pathways whose affiliation is cell type– and altering CD40 À breast cancer cell growth. These in vivo activational state–dependent. These include two subfami- findings illustrated the direct, in vivo immunotherapeutic lies of the stress-activated protein kinases, i.e., JNK (c-jun effect of CD154 on CD40 + carcinomas. However, these N-terminal kinase, also known as stress-activated protein SCID-hu xenograft studies cannot exclude the potential kinase or SAPK) and the p38 mitogen-activated protein contribution of tumor-infiltrating monocyte/macrophages, kinase (MAPK);82 the transcription factors NFkB, activa- which may be activated by CD40 ligation and may produce ting protein-1 (AP-1), nuclear factor for activated T cells antitumor cytokines and/or activated NK cells.66,67 In a (NFAT), signal transducer and activator of transcription recent study with human ovarian cancer cell line and primary (STAT) 3 and 6;82 and other kinases including apoptosis tumor xenografts in SCID mice, rhuCD40L treatment signal-regulating kinase-1 and the germinal center kinase-

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 5 related kinase.83 TRAF1, TRAF2, and TRAF5 also con- increased ICAM-1 expression and IL-6 and IL-8 produc- tained specific DNA binding motifs and can directly regulate tion.54,55,95 Studies with dominant negative TRAF mutants DNA transcription84,85 of CD40-responsive genes including show that TRAF3 was required for CD40-mediated growth members of the TNF (LTa and CD70) and TNF-R family inhibition of various carcinomas,54 whereas TRAF1 and (TNF-R1 and TNF-R2, CD40),82 cell cycle regulatory TRAF3 were required for increased transcription of ICAM-1 proteins Cdk4 and Cdk6,85 antiapoptotic elements (cIAP, and IL-6 in these cell types.54,55 One potential role of inhibitor of apoptosis, and Bcl-xL ), and the proapoptotic TRAF3 may be the modulation of NFkB-dependent path- element DR3, which mediates cell death as well as up- ways.54 However, blockage as well as activation of NFkB regulates CD95/Fas expression.82 It is of interest to note that and the JNK cascade has been reported to be the underlying much of the CD40-mediated phenotypic alterations in B mechanism of CD40-mediated growth inhibition in carci- cells and epithelial tumors can be mimicked through noma cells.17,54,55,62 activation of the EBV-1 encoded latent membrane protein, CD154-induced breast cancer cell growth inhibition LMP-1. This was primarily attributed to LMP-1’s capacity correspondingly activated endogenous apoptotic pathways, to induce binding and oligomerization of TRAF1, TRAF2, as shown by us and others.58,69,75 Whereas CD40 lacks the and TRAF3, and to activate downstream NFkB, AP1 death domain and its link to ICE caspase activation, transcriptional factors, and the c-JNK cascade.17,54,55 apoptosis may nevertheless be initiated through TRAF Thus, unique TRAF subsets are considered to be the association (which in turn recruits death domain containing critical link that converts CD40-initated signals to multi- molecules such as TRADD), through transactivation of other faceted physiologic outcomes. For the normal B cell, TRAF2, TNF-R members that expressed the death domain (such as TRAF5, or TRAF6 recruitment activated the c-JNK path- TNF-R1 and Fas),69,94,95 or by collaterally activating way;86 the extracellularly regulated kinase mitogen-acti- caspases and the JNK pathway.96 These events depended vated protein kinase (ERK-MAP kinase87 ) mobilized on the integrity of the CD40 membrane proximal domain cytoplasmic NFkB to bind multiple gene-regulatory regions but, interestingly, not its TRAF-interacting motif.78 An (kB sites) in the nucleus and resulted in up-regulated increased production of cancer death-inducing cytokines transcription of cytokine (IL-6), adhesion molecule (CD54), such as TNFa, IL-6, IL-8, and granulocyte–macrophage and antiapoptotic proteins such as cIAP1, cIAP2,88 and colony-stimulating factor (GM-CSF),97 and/or corre- 87 94,98,99 Bcl-xL, and A1. The up-regulation of antiapoptotic pro- sponding caspase activation/s may also contribute to teins enhanced the prosurvival effects of CD40 signal- CD40-dependent tumor growth inhibition. ing, whereas coengagement of the membrane Ig receptor In our study of human breast cancer xenografts, we with CD40 limited Fas expression and led to TRAF3- detected significantly elevated proapoptotic bax and bak mediated feedback inhibition of CD40-dependent activation mRNA without alteration of bcl-2 in T47D and BT-20 cells events.84,89,90 For T cells, CD40 binding led to the up- during soluble CD154-induced growth inhibition,51 whereas regulation of TRAF1, which served an antiapoptotic role cotreatment with a CD40L-blocking antibody abrogated much like its involvement in inhibiting antigen-induced cell apoptotic induction and the up-regulation of Bax. Thus, an death in CD8 + T cells.82 CD40 ligation increased c-jun (and altered balance of the bcl-2 family likely contributed to not c-fos) mRNA, leading to SAPK activation45 without CD40-mediated growth inhibition, which may also involve affecting ERK1 and ERK2.45 By comparison, CD40 ligation the endogenous production of cytotoxic cytokines and strongly up-regulated TRAF1 mRNA in human monocytes, autocrine/paracrine induction of cell death.95 and TRAF1 and TRAF5 in the monocytic line THP1. Both normal monocytes and THP1 cells exhibited rapid and transient activation of ERK1/2, their upstream activator, Antitumor immune response activation MAPK kinase (MEK) 1 and 2, as well as the downstream substrate c-Myc.91 Thus, markedly different downstream Both experimental and clinical observations indicate that an signaling processes may account for the diverse physiologic inadequate antitumor immune response may facilitate tumor outcomes that result from CD40 activation in normal B, T, progression. A deficient immune response may stem from and monocytic cells. the lack of tumor immunogenicity (decreased/altered MHC Currently, the molecular mechanisms of CD40-mediated class I expression, decreased and/or heterogeneity of tumor cancer growth regulation remain poorly defined. A signifi- antigen, or lack of adhesion or accessory/costimulatory cant proportion of non-Hodgkin’s lymphomas (NHLs) and molecules), immune unresponsiveness (e.g., tolerance, B-CLL overexpressed TRAF1, whose expression was anergy, immune ignorance, clonal deletion), the presence highly restricted in normal peripheral blood lymphocytes.83 of immunosuppressive proteins (e.g., transforming growth Differences in the expression of combinations of TRAF factor-b, Fas ligand), or changes in the expression of T-cell family proteins may impact their capacity to heterooligo- signal transduction molecules (reviewed in Ref. [100]). merize and recruit proapoptotic proteins to TNF-R mem- CD40 ligation has been shown to stimulate host antitumor bers.83,92 We and others showed that CD154-induced immune response in numerous tumor models. CD40 knock- growth inhibition of human myeloma cells involved the out mice had significantly increased incidence of sponta- up-regulation of TRAF4 and TRAF6 and the induction of neously arising tumors and, unlike mice with a wild-type apoptosis.51,93 For CD40 cross-linking on carcinoma cells, genetic background, could not mount a protective specific the most common molecular outcomes were NFkB activa- immune response against subsequent tumor challenge fol- tion and engagement of the c-JNK cascade, leading to lowing sensitization with GM-CSF–transfected, syngeneic

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 6 B16 melanoma tumor.101 Intratumoral administration of molecules. Ranheim and Kipps123 first demonstrated that agonistic anti-CD40 mAb produced a strong, systemic, activated, CD154-expressing human T cells induced normal tumor-specific cytotoxic T-cell response that was sufficient and leukemic B cells to express B7 and significantly higher to eradicate preexisting CD40 + murine tumors.102 Con- levels of CD54. Follicular lymphomas were highly ineffi- versely, CD154 gene transfer slowed the growth of MCA-1 cient at presenting alloantigens despite their high level of or TS/A murine tumor lines.103 Coimmunization with an MHC expression. For this cell type, CD40 binding up- anti-CD40 cross-linking antibody and the cRL1 tumor regulated B7-1/B7-2 (CD80/86), ICAM-1, and LFA-4, peptide induced cytotoxic response and prolonged survival which may stabilize tumor cell contact with activated T cells of tumor-bearing mice.104 Parental murine bladder cancer through interaction with their respective ligands CD28, MB-49 xenografts were eradicated in 94% of syngeneic LFA-1, and CD2. For Burkitt’s lymphomas, CD40 activa- hosts following injection of CD154-transduced tumor cells, tion resulted in the up-regulation of MHC class I molecules, which protected against subsequent parental, untransfected peptide transporter for antigen presentation (TAP) expres- tumor challenge.105 Immunosensitization contributed sig- sion, and endogenous antigen processing functions, which nificantly to this antitumor approach, as evidenced by the contributed to a more efficient recognition of lymphoma by rapid infiltration of large numbers of CD4 + and CD8 + T specific cytotoxic T lymphocytes. CD40 binding also played cells in the CD154-expressing tumor anlage. a critical role in enhancing the antigen-presenting function The immunostimulatory effects of CD154 correlated with of acute myelogenous leukemia (AML) cells and their its capacity to activate DCs in a number of murine tumor maturation into leukemia DCs.124 –126 AML cells that models.20,106 – 109 CD40 binding produced a prosurvival retained characteristic cytogenetic markers had up-regulated outcome in DCs, attained in part through the activation of the CD80, CD83, CD86 expression, and an increased capacity to antiapoptotic serpin serine protease inhibitor 6 in tumor- stimulate leukemia-specific or allogeneic T-cell cytolytic infiltrating DCs.110,111 Following in vitro maturation treat- activity124 following in vitro treatment with CD40L, GM- ments of GM-CSF and IL-4, CD154 (or TNF) exposure CSF, and IL-4,125 or CD40L and ATRA (all-trans retinoic was required to optimize the antigen-presenting functions of acid).125 Likewise, CD40 cross-linking by recombinant monocyte-derived DCs.112,113 Recombinant CD154 treat- CD154 produced a 6-fold increase in surface MHC class I, ment produced a mature DC morphology, up-regulated Fas, and intracellular adhesion molecule in non small cell expression of adhesion and costimulatory molecules carcinomas that expressed a high level of CD40.59 (ICAM-1, CD83, CD80/86), and heightened secretion of In addition to producing an altered surface phenotype, proinflammatory cytokines (IL-12, IL-6, TNFa) and CD40 binding also increased immunostimulatory cytokine chemokines (IL-8, MIP-1a).113 Activation by CD154 in production in certain tumors. CD40 binding of Hodgkin’s the presence of proinflammatory cytokines or IFNg was lymphoma cells increased the secretion of IL-8, the T-cell– necessary for the ‘‘cross-priming’’ function of DCs, by activating pleiotropic cytokine IL-6, and tumor-regulating which ingested tumor apoptotic body-derived antigens were TNF, which in concert may enhance antitumor immune presented to T cells in the context of HLA class I, leading to response particularly through T-cell attraction and activa- the activation of tumor-specific cytotoxic T effector tion. In addition to significantly increased immunogenicity, 107,114 –117 cells. When exposed to proinflammatory TH1 CD40 ligation of patient acute lymphoblastic leukemia cells antigens, DCs up-regulated CD40 expression and acquired also induced the secretion of the CC chemokines macro- an increased ability to produce IL-12 in response to CD40 phage-derived chemokine (MDC) and thymus and activa- engagement by CD154 + activated T cells.118 IL-12 pro- tion-regulated chemokine (TARC) that serve as potent 119 duction by DCs was pivotal for initiating a TH1 response. chemoattractants for the transendothelial migration of In addition, CD40-activated, monocyte-derived human DCs autologous CCR4 + antileukemia T cells.127 + played a critical role for skewing the CD4 T cell from a TH2 Thus, CD40 stimulation may enhance the effectiveness of 120 phenotype to an IFNg-producing TH1 phenotype. An the host’s antitumor immune response afferently by improv- equally significant role of CD40 binding has also been ing antigen presentation, and efferently by enhancing cyto- identified for initiating an immune response against TH2 toxic activity and cytokine secretion. The critical role of infectious antigens.118,121 In certain experimental models, collateral activation of APC and T cells through CD154– CD154 expression on NK cells may contribute to an CD40 interaction is supported by recent gene transfer studies antitumor outcome. Spontaneous tumor regression of sub- as described in the following section. cutaneously engrafted rat AK-5 histocytomas was mediated by activated CD8 + CD3 À NK cells. The interaction of transplanted, CD40-expressing tumor cells elevated CD154 Induction of antitumor immunity by CD154-transfected expression of NK cells, and ultimately activated NK cells cytotoxicity in part through NFkB mobilization. These findings are consistent with the hypothesis that CD154 Following CD154 transfection, murine lymphoblastic leu- expression by effector cells is regulated by IL-12 and IFNg, kemia A20 cells had up-regulated expression of B7.1, and and an up-regulated CD154 in turn could modulate NK- MHC class I and II molecules. Inoculation of CD154- mediated cytotoxic response.122 transfected cells into a syngeneic host produced a highly Further, CD154 binding enhanced the antigen-presenting effective tumor rejection response that also eliminated capability of cancer cells through an up-regulated expres- preexisting A20 tumor cells at a distant site. This antitumor sion of endogenous tumor peptide and T-cell costimulatory response was mediated by CD4 + and CD8 + T cells and NK

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 7 cells.128 Cancer cells from patients with primary follicular immune-relevant process, producing tumor-specific T cells lymphoma129 and B-CLL130 also had increased expression that were effective in inducing regression of the contralateral of immune costimulatory molecules following in vitro tumor.112 transfection with a CD154 adenovector and a helper virus- Recently, Urashima et al108 utilized a Salmonella free CD154–herpes simplex virus amplicon, respectively. typhimurium vaccine to deliver the CD154 transgene by Both CD154 transgene-expressing lymphoma and CLL the oral route. The live, but attenuated, bacterium penetrated cells have an enhanced capacity to stimulate T-cell the epithelial M cells to reach the Peyer’s patches, and proliferation.129,130 Human NHL or B-CLL cells cotrans- activated local antigen-presenting cells following mucosal fected with adenovectors carrying CD154 or IL-2 produced bacterial uptake mechanisms. This auxotropic S. typhimu- an additive T-cell activation response and sensitized rium strain was nonvirulent because it required metabolites autologous T cells that can recognize parental, untransfected not available in vertebrate tissues for its survival. This oral B-CLL cells.131,132 The toxicity and immunostimulatory gene transfer approach with CD154 resulted in a high degree efficacy of a CD40L and IL-2 expressing B-CLL tumor of protection against the development of a CD40 + B cell vaccine is currently being examined.133 Murine J558 lymphoma (BCL) as defined by significantly prolonged myeloma cells expressing the CD154 transgene activated survival for tumor-bearing mice, and the recruitment of Fas DCs with enhanced B7-1 and ICAM-1 expression, and led ligand-positive TILs. These findings suggest that oral to T-cell–mediated protective immunity against the wild- delivery of CD154 by attenuated S. typhimurium may serve type parental cell line.134 Similarly, human myeloma cells as an effective and safe mucosal immune-activating underwent apoptosis upon introduction of CD154 by an approach against BCL. adenovector, which also induced maturation of bystander DCs that in turn activated tumor-reactive T lymphocytes.135 Thus, CD154 transgenic expression on multiple myeloma CD154-based cancer clinical trials cells can exert a dual effect of direct growth modula- tion50,51,135 as well as stimulating tumor-reactive cytotoxic The efficacy of agonistic CD40-reactive mAb for clinical T lymphocyte activity.136 applications is limited by pharmacokinetic constraints In murine syngeneic solid tumor models, ADV-CD154 particularly for the targeting solid cancers and the likelihood injected directly into established subcutaneous B16 mela- of eliciting human antimouse neutralizing antibodies noma, MCA-1 sarcoma, or CT26 colon carcinoma produced (HAMA) that limit its therapeutic effectiveness. In addition, sustained tumor regression in the majority of tumor-carrying sustained systemic treatment with either CD40-agonistic mice.95,103,109 Systemic immunosensitization can be dem- mAb or the recombinant CD154 protein engenders potential onstrated by virtue of immune rejection of contralaterally toxicity from proinflammatory cytokine production by implanted, nontransfected tumors. Similarly, CD154 gene CD40-activated endothelial cells.18,26,30 Interestingly, this transfer elicited a tumor-specific cytolytic T lymphocyte concern was not realized in 32 patients with advanced solid response that suppressed the growth of established, weakly tumors or intermediate or high-grade NHL, who were treated immunogenic Lewis lung carcinoma in the presence of subcutaneously with recombinant CD154 (rhuCD40L) for IFNg.136 The CD154-activated T-cell response can be 5 days in a recent Phase I clinical trial (Table 3140 ). The transferred to naı¨ve recipients to protect against subse- maximum tolerated dose was found to be 0.1 mg/kg/day, quent tumor challenge.106,136 Preestablished murine K1735 with a serum half-life of 25 hours. Grade 3 or 4 transaminase melanoma, CMT93 colon,109 MB-49 bladder,105 and rat elevations occurred in 14%, 28%, and 57% of patients treated MCA-RH7777 hepatocellular carcinoma137 also underwent at 0.05, 0.10, and 0.15 mg/kg/day, respectively. After a sustained regression following intratumoral injection of single treatment course, 12 patients (38%) had stable CD154-transfected cells. disease, which was maintained in four patients through three According to Loskog et al105 and Nakajima et al,138 additional courses of treatment. Two patients (6%) demon- CD154 transgene expression on tumor cells promoted local strated a clinical response, including one with Stage IV and systemic antitumor immunity by up-regulating B-7 progressive laryngeal cancer who attained a partial response expression on DCs and their secretion of IL-12 that was after one course of 0.1 mg/kg/day rhuCD40L. His remission accompanied by influx of significant numbers of CD4 + , was sustained for 11 additional courses over 12 months, and CD8 + T cells and NK cells at the site of CD154 + tumor cell was then taken off therapy with no further treatment. Three inoculation. The recipients of CD154 transgene-expressing months later, the patient was found to have complete tumor cells withstood subsequent challenge of lethal doses remission and remained biopsy-proven free of disease for of untransfected tumor cells.139 Indeed, this immunosen- a total of 24 months since entered into trial.140 Of the nine sitizing effect of CD154 transgene expression can be NHL patients entered into trial, partial response was attained reproduced with the intratumoral injection of CD154- with one patient with progressive large cell immunoblastic transfected DCs. The CD154-transfected DC most likely T-cell lymphoma after one course of rhuCD40L, as underwent self- and reciprocal CD40 activation and evidenced by regression of skin lesions and reduced presented tumor antigens to naive CD8 + T cells. The in- lymphadenopathy.141 However, the patient did not respond tratumorally introduced CD154 transgene can subsequently to subsequent rhuCD40L treatments. RhuCD40L did not be detected in the spleen of the tumor host, which was produce durable clinical responses in the remaining eight consistent with the conventional expectation of activated DC B-cell NHL patients. These findings indicate that therapeutic migrating into peripheral lymphoid organs to initiate the doses of recombinant CD154 may produce a clinically

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 8 Table 3 Experimental human cancer therapy with CD154

Cancer cell type Approach Clinical observations Putative mechanism References

Solid tumors* and Recombinant CD154 protein, Maximum tolerated dose, Undetermined [140,141] NHL (n=32) subcutaneous, daily Â5 0.1 mg/kg/day; transient liver toxicity, 38% patients attained stable disease; one with partial response, one complete response for 24 months B-CLL (n=11) Autologous, CD154-transfected Significantly reduced leukemic T-cell sensitization by [149,151] leukemia cells, single intravenous cell count and lymph node CD154-transfected bolus injection of 3Â108 –3Â109 size; no long-term toxicity leukemia cells viable cells PCL (n=1) Autologous, CD40-activated PCL Short-term reduction in Adoptive transfer of [152] cells (0.5–1Â108 cells, i.v.), low- circulating PCL cells; no PCL-reactive T cells and dose IL-2 (1.5–3Â106 IU Â19 days); CD154-related side effects tumor cell vaccination autologous, tumor-specific T cells (5.5Â108 –2.5Â109 ) *Stage IV renal cell carcinoma (n=10), head and neck cancer (four), cervical cancer (two), sarcoma (two), rectal carcinoma (one), esophageal carcinoma (one), breast carcinoma (one), peritoneal carcinomatosis (one), adenocarcinoma of unknown primary (one). Sixty-five percent of these patients have been treated previously with chemotherapy and radiation, 39% with a biologic agent.

beneficial effect in selected human cancers, while causing (Table 3). According to Kato et al,148 CD154-transfected only transient hepatotoxicity. patient CLL cells induced an increased level of immune Recent studies have compared the efficacy of the accessory molecules in transfected cells as well as neighbor- recombinant CD154 protein and CD154 gene transfer in ing CD40 + noninfected leukemia cells. In vitro exposure to stimulating host antitumor immune responses.142 Treatment CD154 transgene expressing leukemia cells led to increased with recombinant CD154 or CD154 transgene-expressing serum IFNg and clonal expansion of cytotoxic T lympho- tumor cells was equally effective in inducing the maturation cytes that reacted specifically with autologous, nonmodified of tumor antigen pulsed monocytic DCs. However, CD154 leukemia cells.148 Based on the premise that CD154- transgene-expressing tumor cells may have a more profound expressing tumor cells can provide an immune-stimulatory immunopotentiating effect by virtue of their capacity to effect, which in turn results in altered tumor growth, CLL trigger costimulatory molecule (CD80 and CD86) expres- patients were infused with CD154-transfected autologous 149 sion as well as release of the TH1-activating cytokine leukemia cells in a Phase I dose escalation study. Eleven IL-12.142 Kedl et al143 recently showed that CD40 co- patients with progressive intermediate or high-risk CLL were activation with concomitant tumor antigen presentation can entered into study. A single bolus intravenous infusion of produce a long-term — albeit variable — antitumor out- 3Â108 –3Â109 CD154-transduced CLL cells was well come, whereas CD40 ligation in the absence of tumor tolerated without long-term toxicity and without a maximum antigen actually led to deletion of tumor-antigen–specific tolerated dose. The clinical adverse reactions primarily T cells. Batrla et al144 showed that endogenous CD154 consisted of flu-like symptoms that developed 6–8 hours expression density of activated T cells was down-regulated after infusion and lasted a few days, and mostly likely were following interaction with CD40 + carcinoma cells, suggest- secondary to the induction of endogenous cytokines such as ing that CD40 expression may actually serve as counter- IFNg or IL-12 by CD154-expressing CLL cells.149 The offense by tumor cells to suppress T-cell activation. Thus, infusion of CD154-transduced leukemia cells led to de novo the copresentation of CD154 and tumor-associated antigen or increased expression of immune accessory molecules on through CD154 gene transfer into tumor cells would be the overall noninfected CLL cell population in vivo, including preferred for ‘‘tumor-specific’’ vaccination. The use of ex CD54, CD85, CD80, and CD86 that were detectable for up to vivo CD154-transduced tumor cells serves to limit CD154 4 days postinfusion. In addition, there were rising plasma transgene expression to the tumor microenvironment in view levels of the TH1 cytokines IL-12 and IFNg at 8–48 hours of their homing properties,145 thereby minimizing potential postinfusion in the majority of patients. An increased number toxicity that may result from uncontrolled, systemic CD154 of total circulating T cells and CD4 and CD8 subsets were transgene expression as seen in murine models. CD40L observed in eight of nine patients analyzed. Up to a 2-fold knockout mice developed T lymphoproliferative disorders increase of leukemia-specific T cells was demonstrated by after autologous transplantation of CD154-transfected bone autologous ELISPOT and mixed lymphocyte reactions in all marrow or thymic cells.146 Lethally irradiated normal mice of three patients tested. The observed clinical responses developed Crohn’s disease-like autoimmune bowel disease included significantly reduced leukemia cell counts (>50% at following CD154 transgene expression in the murine 2–4 weeks postinfusion) and up to 90% reduction in lymph thymus.147 node sizes after a single infusion.149 CD40 transduction The clinical applicability of ex vivo CD154-transduced apparently restored the lower basal CD40 expression in CLL tumor has been examined in a number of recent trials cells that was sustained for more than 3 months in some

Cancer Gene Therapy CD154 treatment of human cancer AW Tong and MJ Stone 9 patient. In addition, reconstituted CD40-dependent up- permissive tumor cells that carry the relevant tumor regulation of CD95 was observed, thereby enabling CLL suppressor gene defect (p53, Rb mutation, E2F over- cells to better stimulate CD154 + T cells and increase the expression).155 These late-generation, tumor-selective vec- susceptibility of leukemia cells to cytolytic interaction with tors may facilitate the systemic delivery of the CD154 FasL-expressing T cells.150 This approach may be mimicked transgene, which in turn can potentiate the oncolytic pro- by IL-15, a known T- and NK-cell activator, which also can perties of tumor-selective replicating viruses. also enhance CLL CD40 expression.150 Introduction of CD40-transduced CLL cells also enhanced antibody- dependent cellular cytotoxicity, which augments the activity of antitumor mAb therapy.151 In a recent pilot study, a patient Acknowledgments with plasma cell leukemia (PCL) received a cellular vaccine This work was supported, in part, by the Robert Shanbaum of autologous PCL cells that were previously stimulated with Memorial Fund, the James E Nauss Cancer Research Fund, a CD40L transfectant cell line, low-dose IL-2, and the Summerfield Roberts Grant, the Edward and Ruth Wil- autologous, tumor-specific T cells that have been previously kof Foundation, and the Tri Delta Cancer Research Fund. sensitized with the CD40-activated PCL cells.152 Except for fever and myalgias probably related to IL-2 injections, no other adverse effects were observed. 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