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Gene (2004) 11, 215–226 r 2004 Nature Publishing Group All rights reserved 0929-1903/04 $25.00 www.nature.com/cgt

Role of 4-1BB:4-1BB ligand in cancer Adam TC Cheuk, Ghulam J Mufti, and Barbara-ann Guinn Leukaemia Laboratories, Department of Haematological Medicine, Guy’s, King’s & St Thomas’ School of Medicine, King’s College London, Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK.

The activation of T cells plays a central role in antitumor immunity. In order to activate naı¨ve T cells, two key signals are required. Signal one is provided through the T-cell receptor (TCR) while signal two is that of costimulation. The CD28:B7 molecules are one of the best-studied costimulatory pathways, thought to be the main mechanism through which primary T-cell stimulation occurs. However, a number of molecules have been identified which serve to amplify and diversify the T-cell response, following initial T- cell activation. These include the more recently described 4-1BB:4-1BB ligand (4-1BBL) molecules. 4-1BB:4-1BBL are a member of the TNFR:TNF ligand family, which are expressed on T cells and -presenting cells (APCs), respectively. utilizing the 4-1BB:4-1BBL signaling pathway have been shown to have antitumor effects in a number of model systems. In this paper, we focus on the 4-1BB:4-1BBL costimulatory molecules. In particular, we will describe the structure and function of the 4-1BB molecule, its receptor and how 4-1BB:4-1BBL costimulation has and may be used for the immunotherapy of cancer. Cancer Gene Therapy (2004) 11, 215–226. doi:10.1038/sj.cgt.7700670 Published online 12 December 2003

Keywords: 4-1BB; costimulatory molecules;

n antitumor immunity, cell-mediated responses play a map to murine chromosome 4 at the 75.5 cMposition Icentral role. In order to activate a naı¨ ve , two (Fig 1ai). In mice, the 4-1BB gene spans approximately signals are thought to be requisite. The primary signal or 13 kb consisting of 10 exons, two of them in the 50 signal one occurs through the TCR:MHC:Ag (antigen) untranslated regions and eight in the coding region.11 complex while the second signal or signal two is provided Analysis of the nucleotide sequence of 4-1BB revealed a through costimulation.1 The CD28:B7 pathway (reviewed single open reading frame, which codes for a polypeptide elsewhere2,3) in conjunction with the TCR:MHC:Ag 256 amino acids (a.a.) in length with a calculated stimulus induces high levels of IL-2 production from T molecular mass of 27.5 kDa. The first 23 a.a. was cells. This signaling can provide the essential survival shown to be a signal , followed by a cysteine-rich signals,4–6 preventing the induction of through region, which comprised of four potential TNFR motifs, activation induced cell death (AICD) and the induction of of which the first was partial and the third distinct T cell anergy which occurs in response to signal one from those of the TNFR.11 Almost 30% of the alone.7,8 A number of molecules have been identified a.a. between residues 140–185, which follows the ligand- which function to further enhance and extend the binding domain, were serine or threonines, providing a activation of T cells. Costimulatory molecules includ- potential site for O-linked while a.a. ing 4-1BBL, CD40, OX40L and CD70 have been 186–211 constitute the hydrophobic transmembrane reviewed previously.9 In this report, we focus solely on domain which is followed by the stop-transfer sequence the role of 4-1BB and its receptor 4-1BBL in costimula- containing several basic residues.11 The carboxyl terminal tion and discuss its potential as a target for the part of the cytoplasmic domain contains two short runs of immunotherapy of cancer. three and four acidic residues, respectively, and a sequence of five glycines followed by a tyrosine (Fig 1bi).11 The human homologue of 4-1BB (CD137) was cloned Characterization of 4-1BB gene and from activated human T-cell virus type 1- transformed human T- library.12 Human 4- 4-1BB was first identified in mice by a modified 13 differential screening procedure.10,11 4-1BB was found to 1BB resides on chromosome 1p36 (Fig 1aii), in a cluster of related genes including TNFR2, CD30, OX40 and TAMP/Apo3, which have been shown to be mutated in Received September 9, 2003. several malignancies.14 The human 4-1BB contains Address correspondence and reprint requests to: Dr Barbara-ann 13 Guinn, Leukaemia Science Laboratories, Department of 255 a.a. with two potential N-linked glycosylation sites. Haematological Medicine, Guy’s, King’s & St Thomas’ School of Hydrophobicity analysis predicted that a.a. 1–17 were a Medicine, King’s College London, Rayne Institute, 123 Coldharbour putative signal peptide, which was followed by an Lane, London SE5 9NU, UK. E-mail: [email protected] extracellular domain of 169 a.a. and then a transmem- 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 216 brane region of 27 a.a. between positions 187–213, and 27 kDa15 and was shown to be 60% identical to murine finally a short intracellular domain of 42 a.a (Fig 1bii).13 4-1BB (Fig 1c).13 In the cytoplasmic domain, five regions The molecular weight of the protein was calculated to be of a.a. sequences were conserved between mice and human, indicating that these residues might be important for 4-1BB function.13

Characterization of 4-1BBL gene and its protein

The presence of a ligand for 4-1BB was first confirmed in the EL4 cell line through its binding with a fusion 4-1BB/ Fc protein, the 4-1BBL gene was then cloned through the screening of an EC1 cDNA expression library.16 Murine 4-1BBL consists of 309 a.a. polypeptide. Hydrophobicity analysis predicted that a.a. 83–103 were a signal hydro- phobic domain, while the absence of a signal sequence suggested that 4-1BBL was a type II membrane glyco- protein with an extracellular carboxyterminal domain.16 The 4-1BBL gene maps to murine chromosome 17.16 Human 4-1BBL was first isolated in 1994 by Alderson et al.13 They used a consisting of the extracellular portion of human 4-1BB coupled to the Fc region of human immunoglobulin (Ig) G1 to identify and clone the gene for human 4-1BBL from an activated CD4 þ T-cell clone using a direct expression cloning strategy. Sequence analysis revealed that human 4-1BBL consisted of 254 a.a. and shared a 36% identity with murine 4-1BBL.13 The cysteine residues were not con- served between mice and humans, which raised the possibility that these two genes may in fact represent two distinct ligands for 4-1BB.13 Fluorescence in situ hybridization analyses or FISH analysis showed that the human 4-1BBL gene mapped to chromosome 19 in the region 19p13.3.13 Like murine 4-1BBL was demonstrated to be a type II glycoprotein with a single predicted transmembrane segment.13

Figure 1 Diagrammatic representation of mouza and human 4-1BB chromosomal localizations, structure of their respective and amino-acid sequence homology. (a) The chromosomal location of the 4-1BB gene on (i) chromosome 4 on 4E2 on mice and on chromosome 1p36.2 in humans. In humans and mice the chromo- somal location of 4-1BB is close to other members of the TNFR family including TNFR2, CD30, OX40, and APO3. In humans, the 1p36 region is associated with deletions and rearrangements in several malignancies. (b) Diagrammatic representation of the structure of the 4-1BB protein shown in (i) mice and (ii) humans. Some of the important functional domains of these proteins are illustrated, such as the signal peptide, ligand binding, transmem- brane and intracellular domains as described in references 11 and 13. (c) The a.a. sequence homology between the murine and human 4-1BB molecules is shown in (i) with M4-1BB and H4-1BB representing murine and human 4-1BB, respectively. Differences in the amino-acid sequences between murine and human are indicated in black block and approximately 60% sequence similarity exists between these two species. In (ii) the cytoplasmic tail of murine and human 4-1BB a.a. sequence are shown and conserved regions are indicated; it has been suggested that the cytoplasmic region mediates signal transduction.

Cancer Gene Therapy 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 217 Expression of 4-1BB and 4-1BBL in vivo

4-1BB was found to be induced on CD4 þ and CD8 þ T cells in mice and humans.17–20 The expression in mice takes several hours following stimulation, slowly increas- ing and peaking at 60 hours and declining again by 110 hours.16,19,21 In human, 4-1BB mRNA was detected 1.5 hours after stimulation on T lymphocytes, reaching maximal levels at 8 hours, and declining to background levels by 48 hours.18 In addition to CD4 þ and CD8 þ T cells, 4-1BB was also expressed on activated natural killer (NK),22 dendritic cells (DC)23 as well as neutrophils24 in mice. In humans, the expression of 4-1BB was been reported in follicular DCs,25 ,26 hepatoma cells18 and blood vessels from individuals with malignant tumors.27 The soluble form of 4-1BB was also reported in the serum of patients with rheumatoid arthritis.28 The expression of Figure 2 Diagrammatic representation of the role of the 4-1BB:4- 4-1BB increases in human peripheral blood mononuclear 1BBL pathway in T-cell activation. After capturing antigen, APCs cells after exposure to mitomycin and other DNA- process the intake antigen and present antigenic on their damaging agents, such as , and g- surface. The B7-1, B7-2 and antigenic peptide on APC is through to irradiation.29 be the initial activation signal for naı¨ve T cell. Upregulation of 4-1BB 0 as well as CTLA-4 occurs on the T cell as a consequence of Through the use of 5 deletion constructs of the human activation. In the late immune response or secondary immune 4-1BB promoter in luciferase reporter assays, it was found response, the CD28 is downregulated by the negative feedback that the transcriptional elements mediating 4-1BB upre- receptor CTLA-4. However 4-1BB on T cells, triggered by 4-1BBL on gulation were located in the region between 0.9 and 1.1 kb APC, provides costimulatory signals which sustain B-7:CD28 from the translational start site.30 Characterization of mediated activation and further enhance clonal proliferation and these sites by electrophoretic mobility shift assay and site- the survival of CD8 þ T cell. In the case of CD4 þ , 4-1BB:4-1BBL directed mutagenesis revealed that nuclear factor-kappa B signaling induces clonal expansion, but not the prolonged survival þ (NF-kB) and activating protein-1 (AP-1) were involved, seen in CD8 T cells. and MEK and c-Jun N-terminal kinase-1 (JNK-1) activity were required for activation-dependent 4-1BB upregula- tion. Thus, NF-kB and AP-1 were involved in the TCR very low levels in resting APCs and upregulated upon stimulation-dependent transcriptional regulation of the 4- activation by surface Ig.37 For B7-1, the expression was 1BB promoter.30 predominantly found on B cells and upon stimulation the When comparing 4-1BB with CD28, it becomes expression increased between 24 and 72 hours, however apparent that the expression patterns of these two after 72 hours, the expression declined rapidly.37 B7-2 was costimulatory molecule receptors were very different in upregulated more rapidly on both professional APCs and T cells. CD28 was expressed on all murine T cells and on lymphocytes but had a similar peak expression time the majority human T cells.7,8 However, upon activation between 24 and 96 hours.37 Although the B7 and 4-1BBL of T cells, a dramatic increase in CD28 expression was costimulatory molecules were inducible, 4-1BBL reached reported in terms of both the mRNA and protein levels.31 its peak expression within a shorter time following CD4 þ CD25 þ regulatory T cells express 4-1BB constitu- stimulation. tively,32,33 while 4-1BB expression was induced from undetectable levels on naı¨ ve cells, adding credence to the suggestion that 4-1BB expression was a result of the Role of 4-1BB:4-1BBL in primary immune responses upregulation of the B7 costimulatory molecules through CD28 signaling (Fig 2).20 The existence of 4-1BBÀ/À and 4-1BBLÀ/À knockout 4-1BBL was found to be expressed following stimula- mice38,39 has allowed a number of investigations into the tion on professional APCs including DCs and macro- necessity of 4-1BB:4-1BBL in immune responses. Kwon phages as well as activated B cells in both human and et al39 showed that T cells from 4-1BBÀ/À mice had mice.13,16,21,23,34 Human 4-1BBL message was detected as enhanced proliferation in response to mitogens, or a- early as 30 minutes following stimulation through CD3. However, the T-cell immune responses of 4-1BBÀ/À immobilized CD3 monoclonal (mAb) and peaks mice, as measured by production and CD8 þ T- at 1 hour.13 4-1BBL was also present at high levels in the cell cytotoxic T (CTL) activity, were dimin- sera of some patients with hematological diseases35 as well ished. In 4-1BBLÀ/À mice, Bertram et al38 showed that in as on some cell lines.36 the initial expansion of Db/NP366-374-specific CD8 þ T The inducible expression pattern of 4-1BBL was similar cells in response to influenza virus infection, 4-1BBLÀ/À to that of B7-1 and B7-2 in the CD28:B7 pathway. The mice showed a decrease in virus-specific T cells late in the costimulatory molecules B7-1 and B7-2 were expressed at primary response.

Cancer Gene Therapy 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 218 In mice, it has been shown that in conjunction with CD4 þ T-cell subpopulation in vivo with mAb resulted in ‘‘signal one’’, the interaction of 4-1BBL with 4-1BB the loss of the antitumor effect.47 CD4 þ T cells may not provides costimulatory signals to both CD4 þ and CD8 þ be required during the generation of the initial immune T cells through the activation of NF-kB, c-Jun and p38 response but may be important in the generation or downstream pathways (Fig 3).40 Although stimulation of maintenance of long-term memory responses.48 It was the 4-1BB:4-1BBL signaling pathway leads to both CD4 þ found that the expansion and cytolytic function of tumor and CD8 þ T-cell activation,19,41,42 evidence has shown reactive CD8 þ T cells was enhanced by CD4 þ T cells.49 that it caused the preferential expansion of CD8 þ T cells. Apart from T cells, other cells in the Shuford et al43 used mAbs to show that 4-1BB signaling have been shown to be influenced by 4-1BB:4-1BBL markedly enhanced -g production from CD8 þ during immune responses. Crosslinking of 4-1BBL on T cells and that the a-4-1BB mediated proliferation of by 4-1BBFc plus a-Fc stimulates the CD8 þ T cells appeared to be IL-2 independent. Use of a- production of IL-6, IL-8 and TNF-a but not IL-12.50 4-1BB Ab, in vitro, to stimulate CD8 þ T cells with The engagement of 4-1BBL, in the context of a-IgM downregulated CD28 showed that CD28 expression was providing signal 1, relays costimulatory signals to restored, while expression of the memory marker stimulate B-cell proliferation.21 Recent studies using CD45RO and CC chemokine receptor 6 (CCR6) and transgenic mice expressing 4-1BBL under control of the content of were also enhanced. Lee et al44 MHC II I-Ea promoter showed that they developed showed that 4-1BB promotes the survival of CD8 þ T progressive splenomegaly and selective depletion of lymphocytes by increasing expression of antiapoptotic B220 þ B cells accompanied with low levels of circulating genes bcl-XL and bfl-l via 4-1BB-mediated NF-kB IgG. In addition, these mice exhibited defective humoral activation preventing AICD. As 4-1BB triggering could responses to Ag challenge suggesting that 4-1BBL may be protracted from the TCR signal, 4-1BB agonists may regulate humoral immune responses through the control function through these mechanisms to enhance or rescue of the survival of .51 suboptimal immune responses.45 The evidence suggests Selective depletion of NK cells in mice using mAb that regulatory signals delivered by the 4-1BB receptor completely abrogated the antitumor effect in various play an important role in the regulation of CD8 þ T cells animal tumor models,22,52 indicating that NK cells may in cellular immune responses to Ag. 4-1BB also plays a modulate either immunoregulatory or effector function in marked role in the differentiation of effector memory response to 4-1BB:4-1BBL signaling. Activation of 4-1BB CD8 þ T cells.46 Although the 4-1BB signaling pathway in the abrogates GM-CSF-mediated was shown to lead to a preferential stimulation of CD8 þ anti-apoptosis, which may be associated with massive compared with CD4 þ T cells, selective depletion of neutrophil accumulation and consequent tissue damage.24 Triggering of the 4-1BB receptor in human monocytes was able to activate IL-8 and TNF-a production whereas it inhibited IL-10 production. More recently a new function for 4-1BB as a DC activator was observed. 4-1BB was shown to induce IL-6 and IL-12 production as well as upregulate the costimulatory molecules B7-1 and B7-223 upon receiving stimulus from adjacent APC or tumor cells.23,53,54

Role of 4-1BB:4-1BBL in secondary immune responses

Most of the evidence of the involvement of 4-1BB:4-1BBL in secondary immune responses has been reported following studies of viral infections in mice. BALB/c mice immunized with influenza strain X-31 were sacrificed 3 weeks after treatment and the splenocytes were isolated. The cells were then restimulated in vitro with the H-2d B Figure 3 Diagrammatic representation of 4-1BB:4-1BBL-mediated lymphoma, K46J, expressing 4-1BBL and the influenza signaling events at the cell surface. Two signals are necessary to virus peptide. Blockade of the 4-1BB:4-1BBL signaling activate the naı¨ve T cell, the first is through TCR:MHC:Ag, while through the inclusion of a soluble form of the 4-1BB signal two involves the costimulatory signals (e.g. CD28:B-7, 4- receptor during this in vitro restimulation partially 1BB:4-1BBL). Adhesion molecules (eg. CD40:CD40L, ICAM-1:LFA- abrogated the secondary CTL responses.55 Using LCMV, 1, LFA-3:CD2) are important during the interaction as they are either 4-1BBL-deficient or wild-type mice were immu- thought to extend the period of time during which the two cells are nized with a (LCMV) peptide NP396-404. When agonistic held close together. Upon trigging of 4-1BB receptor and TCR, NF- þ kB, c-Jun, p38 downstream pathway are activated which activate a-4-1BB Ab was given, the CD8 T-cell responses in 4- cytokine production and secretion. Activation of NF-kB also 1BBL-deficient mice were augmented to levels similar to promotes the survival of CD8 þ T lymphocytes by increasing those in wild-type mice immunized with the same agent.

expression of antiapoptotic genes bcl-XL and bfl-l. Upon challenge, wild-type mice still had -specific

Cancer Gene Therapy 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 219 T cells and were protected against viral infection, CTLA-4 signaling.63 A number of studies have demon- demonstrating that peptide vaccination can induce long- strated effective antitumor targeting in clinical trials using term protection. However, 4-1BBLÀ/À mice were not the costimulatory molecules but to date these have solely protected from the infection56 suggesting that the 4- utilized the B7:CD28 pathway (Table 1).64–68 1BB:4-1BBL pathway was crucial for the development of Autoimmune disease may be initiated when T cells are the secondary immune responses. inadvertently activated to respond to normal tissues The latter timing of 4-1BB expression following T-cell perhaps due to concurrent viral infection. Evidence of activation suggests that 4-1BB:4-1BBL may be important the ways in which viruses can mis-direct the immune in the late immune response when the CD28:B7 signaling system and lead to autoimmune disease in humans are is dampening. However, signals through 4-1BB:4-1BBL discussed in detail elsewhere.69 In immunotherapy, a have been shown to be involved in the regulation of balance has to be reached in which CD8 þ T cells are proliferation and survival of lymphocytes and appears to stimulated to kill infected or malignant cells but are not be able to work in both a CD28 independent and inadvertently stimulated to recognize normal tissues and dependent manner.57,58 In the absence of CD28, 4-1BB cause the autoimmune destruction of healthy organs. molecules have been able to prevent AICD and rescue and Myers et al70 showed that 4-1BB and Toll-like receptor sustain ongoing immune responses.58,59 led to the survival of specific effector CD8 þ T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and preventing autoimmune disease. Although, Can the 4-1BB:4-1BBL pathway work in a CD28 immunotherapy protocols seek to avoid the induction of dependent manner? normal tissue destruction there are occasions when relatively mild side effects such as vitiligo, opportunistic The B7:CD28 costimulatory pathway seems to be infections and EBV-related lymphoproliferative disease essential for the induction of 4-1BB, whereas 4-1BB do occur. However these autoimmune-related side effects expression provides its own positive feedback loop. The are treatable and are generally considered to be a CTLA4 receptor limits T-cell-mediated immune responses reasonable trade-off71 for the removal of a life-threaten- while together the CD28 and 4-1BB pathways facilitate 58,59 ing tumor. both T-cell survival and effector cell development. In the remainder of this paper, we will focus solely on We found that 4-1BBL can enhance antitumor re- some of results of preclinical studies using 4-1BB:4-1BBL sponses in the absence of CD28, however for protective as a vehicle to enhance antitumor responses (Table 2). immunity, CD28:B7 signaling was necessary.60,61 We demonstrated that using the A20 B-cell lymphoma model in CD28À/À mice, 4-1BBL enhanced antitumor responses Adoptive transfer of ex vivo costimulated T cells even in the absence of CD28; however, the CD28:B7 One strategy has involved the isolation of T cells from pathway was required for long-term protective immunity tumor-bearing mice which were expanded, stimulated ex against the parental A20 tumor. These results support the vivo and transferred back into tumor-bearing mice. idea that different immune responses vary in their Recently, this approach was used by Strome et al,72 dependence on costimulation and suggest a role for 4- who isolated T cells from the tumor-draining lymph node 1BBL in augmenting suboptimal CTL responses in vivo. of mice bearing disseminated micrometastasis of a poorly immunogenic, MHC class I-negative A9P . The T cells were stimulated through both the CD28 and 4-1BB 4-1BB:4-1BBL in tumor immunogene therapy pathways and then adoptively transferred into another mouse with A9P melanoma. Results showed that a 60% The aim of tumor immunotherapy has been to generate cure rate was achieved. long-lasting functionally active CD8 þ T cells specific for In a murine model tumor-draining lymph the tumor cells. Tumors that develop are thought to have nodes (TDLN) were isolated from mice bearing MCA205 escaped immune surveillance. One reason for this belief is tumors. The cells were then stimulated in vitro using a- that patients with immunosuppressive diseases such as CD3, a-CD28 and/or a-4-1BB Ab and returned to AIDS tend to develop tumors at an increased frequency. syngeneic mice bearing MCA205 tumors. The number Tumors may evade the immune system in one or more of of pulmonary metastases in the recipient mice were the following ways: through (1) low-level expression of significantly reduced and survival was prolonged in mice the major histocompatability complex (MHC) I mole- which received a-41BB mAb compared with those that cules; (2) poor costimulatory molecules expression; (3) did not.73 These studies demonstrated that the adoptive absence of recognizable tumor Ags; (4) the secretion of transfer of ex vivo-stimulated T cells may increase their immunosuppressive substances such as FasL. A more potential for cancer immunotherapy in some pre-clinical thorough review of these can be found elsewhere.62 In animal studies. order to activate T cells against the tumor, strategies must However when applied in humans, adoptive therapy be developed that overcome the ways in which tumors using T cells has some limitations. It has been difficult to escape the immune system or the ways in which the generate sufficient T cells ex vivo to transfer back into immune system limits it’s response such as through patients. The functional capacity of CD8 þ T cells has

Cancer Gene Therapy 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 220 been shown to be altered following the in vitro T-cell

68 69 70 71 72 culturing phase. Even when a large number of functional T cells were generated and transferred into the patient, the adoptive therapy was still unsuccessful by virtue of the range of mechanisms by which tumors evade the immune system. Details of the problems of adoptive transfer of T cells are reviewed in more detail else- where.74

Therapy using a-4-1BB Ab Another strategy has been to stimulate T cells to target tumors through the injection of Ab into the host. The in vitro hypothesis behind this was that if the tumor did not have sufficient costimulatory molecule expression then injecting mAb against 4-1BBL, which binds to 4-1BB on the T cell, would activate the tumor-specific T cell, and lead to tumor cell lyses. Melero et al47 showed that the intraperitoneal (i.p.) injection of a-4-1BB mAb could eradicate large estab- lished tumors in mice, including the poorly immuno- genic Ag104A and the highly tumorigenic P815 Nine patients had measurableresponse; disease; two two patients patients had had stable partial disease Three patients experienced clinically stable disease All patients had evidenceCEA of expression leukocytic in infiltration vaccine and had biopsy stable sites. disease Six after patients four vaccinations Patients who received COACTsof secreted IFN higher gamma amounts andanti-CD3/anti-CD28 GM-CSF restimulation on Nine patients were evaluable6 for weeks restaging after examinations theof antibody them application, (22%) with showing two a local clinical response mastocytoma. The immune response induced by a-4- 1BB mAb was mediated by both CD8 þ and CD4 þ T cells and was accompanied by a marked augmentation of tumor-specific CTL activity. Treatment of 3-day intracranial MCA 205 sarcoma or in vitro GL261 glioma with mAb against 4-1BB (3E1 and 1D8) given i.p. resulted in the cure of four and two of five of the MCA205 sarcoma-bearing mice. Treatment of mice with either mAb led to prolongation of survival and cure of disease in two of each of the five mice harboring a GL261 glioma.75 It was also noted that the successful treatment of intracranial tumors induced concomitant regression of subcutaneous tumors. However, no anti- tumor effects against the poorly immunogenic B16/D5 melanoma were observed. In an orthotopic model of metastatic colon carcinoma and transferred into patient,administered systemic as IL-2 an was adjuvant to express both humanand carcinoembryonic B7-1. antigen Three cohortswith of advanced six CEA-expressing patients, adenocarcinomas, each were treated with increasingpoxvirus doses vaccine of the canary antigen (CEA) were immunizedcanary with pox a virus thatengineered has to been encode thefor gene the CEA and B7-1 (anti-CD28)-coated beads (COACTs) in the presence of .were The then coactivated transferred T into cells patient bispecific and CD28 established in the liver of mice, the authors generated an effective tumor-specific CD8 þ T-cell response through a combination of IL-12 gene therapy and systemic i.p. delivery of an agonistic mAb against 4-1BB. In the IL-12 No. of patients Status/comments Outcomeplus a-4-1BB combination Reference treatment, the innate and adaptive antitumor immune responses were synergistic, as animals bearing simultaneous hepatic and multiple pulmonary metastases were quantitatively cured of their

Stage of trial diseases. Both NK and CD8 þ T cells were necessary in maintaining the long-term antitumor immunity, as depletion of either cell type in the cured animals abolished their abilities to reject tumor cells implanted at distal sites.76 In a systemically delivered murine JC breast cancer Costimulatory molecules used CD28 Phase I 10 Coactivated T cells with OKT3 (anti-CD3) and 9.3 treated with a-4-1BB mAb i.p. there was regression of pre-established tumors and a survival rate of 87%. The group also compared the systemic agonistic Ab with local 4-1BBL gene delivery in combination with IL-12.

Selected clinical trials involving cancer immunotherapy using the CD28 costimulatory pathway They found that the latter method was also able to eradicate established tumor with a similar survival rate of 78%. However, only survivors who had received B7-1 Phase I 15 Tumor was transduced with B7-1 gene Table 1 Cancer type Adenocarcinoma B7-1Adenocarcinoma Phase I B7-1Renal cell, ovarian, breast, and 6colorectal Phase IB-cell lymphoma Nonreplicating canary pox virus was constructed 39 CD28treatment Patient with tumor expressing carcinoembryonic Phase I with the 10 IL-12 Patients were treated by injection of CD3xCD19 and 4-1BBL genes showed

Cancer Gene Therapy Table 2 Summarized details of murine antitumor models which target the 4-1BB:4-1BBL pathway Stragery Cancer type Comments Outcome Reference

Adoptive transfer Melanoma A9P T cells from tumor bearing mice were 60% cure rate was achieved 72 of ex vivo costimulated stimulated in vitro through CD28 and T cell 4-1BB pathway and adaptively transferred into mice bearing A9P tumor Fibrosarcoma MCA 205 T cells from tumur-draining lymph nodes were Pulmonary metastases significantly 73 costimulated ex vivo using reduced, survival was prolonged a-CD3, a-CD28 and a-4-1LL Ab, and transferred into syngeneic mice bearing MCA205 tumor

Therapy using Sarcoma Ag104A a-4-1BB Ab was injected i.p. Large established tumor was eradicated 47 a-4-1BB Ab Mastocytoma P815 a-4-1BB Ab was injected i.p. Large established tumor was eradicated 47 Fibrosarcoma MCA 205 a-4-1BB Ab was injected i.p. Four and two of groups of five mice were cured 75 Glioma GL261 a-4-1BB Ab was injected i.p. Prolongation of survival and cure of disease in 75 two of five mice Metastatic colon carcinoma IL-12 plus a-4-1BBL Ab were used Animal bearing simultaneous hepatic and multiple 76 pulmonary meastases were quantitatively cured Breast cancer JC Local 4-1BBL gene delievery in It was able to eradicate established tumour with a 77 combination with IL-12 survival rate 78%

Whole cell vaccine Mastocytoma P815 P815 cells were transduced with 4-1BBL gene Developed long-term immunity against wild-type tumor 78

and injected into mice Cheuk immunotherapy ATC cancer in ligand 4-1BB:4-1BB Sarcoma Ag104A Ag104A cells were transduced with 4-1BBL gene Developed long-term immunity against wild-type tumor 78 and then injected into mouse B-cell lymphoma A20 Mice injected with 4-1BBL expressing A20 No tumors were formed for the 150 days follow-up period 81

Squamous cell 4-1BBL cDNA was first induced into NRS1 cells Syngeneic mice acquired specific immunity 82 al et carcinoma NRS1 against wild-type tumor T-cell lymphoma EL4 4-1BBL was transfected into EL4 cells, cells It was able to induce tumor regression 83 were then injected into mice Colon cancer MCA26 MCA26 cells was first injected into the liver. The survival were improved than the control group 52 Adenoviral vector was used to transduce IL-12 and 4-1BBL DNA into the established liver tumor Liver metastases of Intratumor adenoviral mediated gene Survival rate was 72% 52 breast cancer transfer of the 4-1BBL Melanoma K1735 Single chain of Fv fragments of a a-4-BB mAB Vaccinated mice rejected established 84 gene was transduced into the tumor cells wild-type tumor acrGn Therapy Gene Cancer 221 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 222 significantly potent, systemic and tumor-specific T-cell- Introduction of 4-1BBL cDNA into NRS1, a mediated immunity.77 murine squamous cell carcinoma, efficiently elicited It would appear that the transfection of 4-1BBL cDNA antitumor immune responses in syngeneic mice which into tumor cells to make a whole cell tumor vaccine may acquired specific immunity against wild-type tumor.82 not be as effective at inducing antitumor responses when T-cell depletion studies showed that CD8 þ , but not compared to the use of a-4-1BB mAb. In the murine CD4 þ T cells were essential for tumor eradication. In sarcoma cell line Ag104, the tumor cells which express 4- addition to B7-1 and B7-2, the host-derived 4-1BBL was 1BBL had no therapeutic activity unless they were also also found to be involved in the secondary antitumor transfected with B7-1.78 However, the injection of a-4- responses.82 1BB Ab into mice with the same tumor (Ag104) caused Xiang,83 transfected 4-1BBL into the moderately tumor destruction.47 In addition mAb against the T-cell immunogenic murine T-cell lymphoma EL4 cell line and activation molecule 4-1BB was effective in the treatment the poorly immunogenic, highly metastatic to the lung, of established mouse tumors. In the Ag104 tumor model, variant of BL6 melanoma cell line called BL6-10. 4-1BBL cDNA did not engage antitumor immune Expression of the costimulatory molecule 4-1BBL was responses without the help of CD28:B7 signaling, thus, able to induce tumor regression of the modified EL4/4- using mAb can provide a more effective antitumor 1BBL but not BL6-10/4-1BBL tumor cell lines in immune response even in the absence of CD28:B7 syngeneic BALB/c mice. The tumor regression was mainly signaling. However mAbs may still not provide the better mediated by CD8 þ T cells that led to protective immunity treatment as 4-1BB mAbs have been shown to abrogate against the parental EL4 tumor. The result reaffirmed the T-cell-dependent humoral immune responses in vivo antitumor potential of engineered tumor cells expressing through the induction of helper T-cell anergy.79 Moreover the costimulatory molecule 4-1BBL, especially in combi- in a personal communication by Dr R Mittler (Depart- nation with other costimulatory molecules such as B7-183 ment of , Emory University School of Medicine, and/or B7-281 in cancer vaccines. Atlanta, Georgia 30329, USA) quoted by Hellstrom and In a colon cancer model, syngeneic BALB/c mice Hellstrom,80 mAb may induce or worsen autoimmune received an intrahepatic injection of the poorly immuno- diseases that were mediated by CD8 þ and/or CD4 þ T genic MCA26 colon cancer cells.52 Various combinations cells. of replication-defective adenoviruses expressing IL-12 and 4-1BBL cDNA were injected into the established liver tumor. Changes in tumor size and animal survival were Whole cell vaccines created using 4-1BBL then monitored. The long-term survival rate of mice The most common immunotherapeutic use of 4-1BBL to treated with the combination of IL-12 and 4-1BBL were date has been the modification of tumor cells with 4- significantly improved over that of animals in the control 1BBL cDNA for whole cell vaccinations. The theory groups. In vivo depletion of NK cells or CD8 þ T cells behind this treatment is that if a tumor cell already completely abolished the long-term survival advantage of expresses a medley of mostly undefined tumor Ags then the IL-12 plus 4-1BBL-treated animals. The systemic the modification of the tumor cell to improve its ability to immunity induced by this combination treatment pro- act as an APC, would avoid the need to define these Ags, tected animals against a subcutaneous challenge with while costimulatory molecules transfection would provide parental MCA26 cells. Adenovirus-mediated transfer of an abundance of signal two. IL-12 and 4-1BBL cDNAs directly into liver tumors Mice inoculated with murine P815 mastocytoma or resulted in tumor regression that required both NK and A104A sarcoma cell lines expressing 4-1BBL develop a CD8 þ T cells and generated a potent, long-lasting strong CTL response and long-term immunity against antitumor immunity. wild-type tumor. The optimal effect of 4-1BBL in CTL In a murine liver metastases of breast cancer model, stimulation required B7-CD28 signaling since Ab block- intratumor adenoviral-mediated gene transfer of the 4- ade of this downregulated the expression of 4-1BB on T 1BB led to regression of a pre-established tumor and the cells and decreased CTL activity.78 survival rate was 78%.52 In studies using the A20, B-cell lymphoma cell line,81 An alternative method for the creation of a whole cell mice injected with tumors expressing the vector backbone vaccine was the transfer of cDNAs encoding fragments of alone (A20/CMV) or B7-1 (A20/B7-1) developed tumors mAb against 4-1BB. This vaccine appeared to stimulate within 25 days of subcutaneous injection. In contrast, the immune system in a similarly efficacious manner to a- mice injected with A20/4-1BBL were tumor free for the 4-1BB mAbs. Ye et al,84 constructed a vector encoding 150-day follow-up period, and few (25%) of the mice cell-bound single-chain Fv fragments from 1D8 a-4-1BB. injected with A20/B7-2 developed tumors. Almost all They transfected the vector into K1735 melanoma cells mice which resisted the initial tumor challenge rejected a which expressed low levels of MHC class I molecules and later systemic challenge with the parental tumor cell line. were poorly . The transfected cells in- Tumorigenicity experiments using nude mice indicated the duced a strong type 1 T-helper cell response, for which requirement for T cells for variant rejection while CD4 þ but not CD8 þ T lymphocytes were necessary and Ab blocking studies indicated that both CD28 and this response also involved NK cells. Vaccinated mice 4-1BB signaling were involved in CTL-mediated tumor rejected established wild-type K1735 tumors growing as lyses ex vivo. subcutaneous nodules or in the lung.

Cancer Gene Therapy 4-1BB:4-1BB ligand in cancer immunotherapy ATC Cheuk et al 223 There are technical difficulties associated with whole cell that 25% of mice injected with A20 lymphoma cells vaccine protocols, cells are very difficult to expressing low levels of 4-1BBL developed tumors in the culture and transfect ex vivo and it has been difficult to 150 days post-injection follow-up period but no tumors obtain a large enough gene-modified population for developed in mice injected with A20 expressing moderate transplantation back into patients.85 Most whole cell or high levels of 4-1BBL. Hence, for the development of a vaccine models in mice have involved injecting the modified more effective antitumor vaccine, a more discerning live tumor cells into mice in the first instance and then strategy might involve the modification of the tumor cell challenging with primary tumor cells. From the lack of to express several different costimulatory molecules each publications to the contrary, and our own unpublished with a high level of expression. experiences, it would appear that eliminating established tumors using whole cell vaccines is also very difficult. Conclusion

Capacity of the costimulatory molecules to enhance In animal studies, 4-1BB:4-1BBL seems to give very antitumor responses promising results in anti-tumor therapy and has provided us with a lot of information regarding the basic biology of It has not been easy to compare the efficacy of different costimulation. However, there appears to be a long way costimulatory molecules in the various tumor immu- to go for this to apply to human clinical trials. Many notherapy protocols, as different tumors have an indivi- technical problems still need to be solved and continuing dual molecular signature of gene expression. This efforts in animal models and human clinical trials will signature varies within the cells of the tumor and with help provide the information necessary to improve tumor progression. To date only a small number of therapies in the future. studies have directly compared the costimulatory mole- cules within a single tumor model system.78,81 These studies have shown that transfection of 4-1BBL cDNA Acknowledgments into a tumor cell which had innate B7-1 and B7-2 expression could create a whole cell tumor vaccine which We would like to thank Professor Farzin Farzaneh for his was more efficacious than transfection of the same cells suggestions and the Departments of Haematological with either B7-1 or B7-2 alone. Medicine and Molecular Medicine for their support. BG In general, 4-1BB receptors were only inducible on is funded by the Leukaemia Research Fund. activated T cells and the costimulatory pathway, B7/ CD28 appeared to be necessary to activate T cells through the primary T-cell activation or secondary responses in 4- References 1BB knockout mice.38,39 4-1BB:4-1BBL acts as an amplifier of the existing costimulatory signals but 1. Lafferty KJ, Cunningham AJ. A new analysis of allogeneic B7:CD28 was still the primary and generally the most interactions. Aust J Exp Biol Med Sci. 1975;53:27–42. effective mode of initiating the immune response (at least 81 2. Carreno BM, Collins M. The B7 family of ligands and its in antitumor responses). Coexpression of 4-1BBL and receptors: new pathways for costimulation and inhibition of B7-1 in the poorly immunogenic AG104A sarcoma immune responses. Annu Rev Immunol. 2002;20:29–53. enhanced the induction of effector CD8 þ T cells which 3. Chambers CA, Allison JP. Costimulatory regulation of T could reject even the poorly immunogenic wild-type cell function. Curr Opin Cell Biol. 1999;11:203–210. AG104A tumor while neither 4-1BBL nor B7-1 single 4. Tai XG, Toyooka K, Yashiro Y, et al. CD9-mediated transfectants were as effective.78 NRS1 was a murine costimulation of TCR-triggered naive T cells leads to squamous cell carcinoma that constitutively expresses B7- activation followed by apoptosis. J Immunol. 1 at high levels, yet it was the introduction of the 4-1BBL 1997;159:3799–3807. 5. Yashiro Y, Tai XG, Toyo-Oka K, et al. A fundamental cDNA that efficiently elicited antitumor immune re- difference in the capacity to induce proliferation of naive T sponses in syngeneic mice which then acquired specific 82 61 cells between CD28 and other co-stimulatory molecules. Eur immunity against the wild-type tumor. Guinn et al J Immunol. 1998;28:926–935. showed that in a A20 model in mouse, protective 6. Zuckerman LA, Pullen L, Miller J. Functional consequences immunity against parental tumors involved both the 4- of costimulation by ICAM-1 on IL-2 gene expression and T 1BBL and CD28 pathways. All these data suggested a cell activation. J Immunol. 1998;160:3259–3268. synergistic effect between the B7:CD28 and 4-1BB:4- 7. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system 1BBL costimulatory pathways. B7/CD28 pathway does of T cell costimulation. Annu Rev Immunol. 1996; not always protect the host against wild-type tumor but 14:233–258. the B7/CD28 pathway was required for the optimal effect 8. Chambers CA, Allison JP. Co-stimulation in T cell 78 responses. Curr Opin Immunol. 1997;9:396–404. of 4-1BB:4-1BBL. 9. Watts TH, DeBenedette MA. T cell co-stimulatory mole- The level of 4-1BBL expression on modified tumor cells cules other than CD28. Curr Opin Immunol. 1999; also appears to be important. Higher levels of the 11:286–293. costimulatory molecules appeared to be more effective 10. Kwon BS, Weissman SM. cDNA sequences of two inducible in inducing antitumor responses. Guinn et al61 showed T-cell genes. Proc Natl Acad Sci USA. 1989;86:1963–1967.

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