Role of 4-1BB:4-1BB ligand in cancer immunotherapy

The activation of T cells plays a central role in antitumor immunity. In order to activate na?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 antigen-presenting cells (APCs), respectively. Therapies 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.     

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.

To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-zeta-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.     

Divergent effects of 4-1BB antibodies on antitumor immunity and on tumor-reactive T-cell generation

4-1BB is an inducible receptor-like protein expressed rapidly by both CD4 and CD8 T-cells after activation. 4-1BB cross-linking, either by binding to 4-1BBL or by antibody ligation, delivers a costimulatory signal to enhance T-cell activation and proliferation. Previous studies have demonstrated that the administration of 4-1BB monoclonal antibodies (mAbs) induces antitumor immune responses. In the current study using several murine tumors, we examined the systemic effects of 4-1BB mAb on the growth of s.c., intracranial (i.c.), and pulmonary metastases. In addition, the effects of 4-1BB mAb on the generation of antitumor effector T cells were examined. Treatment of 3-day i.c. MCA 205 sarcoma and GL261 glioma with the antibody resulted in prolongation of survival and cure of disease in some mice, whereas only minimal therapeutic effects were observed in established s.c. and pulmonary tumors. No antitumor effects against the poorly immunogenic B16/D5 melanoma were observed. Interestingly, successful treatment of i.c. tumors induced concomitant regression of s.c. tumors. Experiments using severe combined immunodeficient mice and mice depleted of either CD4 or CD8 T cells demonstrated T-cell dependence of the antitumor effects. For generation of effector T cells in the tumor-draining lymph nodes (LNs), administration of 4-1BB mAb had adverse effects, despite the apparent hypertrophy of the LNs. During in vitro activation of tumor-draining LN T cells with anti-CD3 and interleukin 2, the 4-1BB mAb augmented proliferation, resulting in an increase in CD8 T cells. However, they were less therapeutic than not treated LN cells. In adoptive immunotherapy, the coadministration of 4-1BB mAb enhanced the therapeutic efficacy. These results thus demonstrate the limits and potential advantages of 4-1BB antibody interactions with antitumor T cells in vivo and in vitro and suggest that therapeutic interactions of the antibody may be used in a variety of immunotherapeutic approaches.     

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. EXPERIMENTAL DESIGN: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. RESULTS: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype. CONCLUSIONS: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.     

Absence of class II-associated invariant chain peptide on leukemic blasts of patients promotes activation of autologous leukemia-reactive CD4+ T cells

Immune escape in cancer poses a substantial obstacle to successful cancer immunotherapy. Multiple defects in HLA class I antigen presentation exist in cancer that may contribute to immune escape, but less is known about roles for HLA class II antigen presentation. On class II(+) leukemic blasts, the presence of class II-associated invariant chain peptide (CLIP) is known to be correlated with poor survival in acute myeloid leukemia (AML). In this study, we evaluated the functional significance of CLIP expression on leukemic blasts of AML patients. CD4(+) T cells from patients were cocultured with autologous CLIP(-) and CLIP(+) primary leukemic blasts and analyzed for several functional parameters by flow cytometry. Increased HLA-DR and IFN-γ expression was observed for CD4(+) T cells stimulated with CLIP(-) leukemic blasts, in contrast to CLIP(+) leukemic blasts, which indicated an activation and polarization of the CD4(+) T cells toward T-helper 1 cells. In addition, CLIP(-) leukemic blasts induced greater outgrowth of effector memory CD4(+) T cells (with HLA-DR-restricted T-cell receptor Vβ repertoires) that were associated with better leukemia-specific reactivity than with CLIP(+) leukemic blasts. Our findings offer a clinical rationale to downmodulate CLIP on leukemic blasts as a strategy to degrade immune escape and improve leukemia-specific T-cell immunity in AML patients.     

A functional anti-human 4-1BB ligand monoclonal antibody that enhances proliferation of monocytes by reverse signaling of 4-1BBL

4-1BB Ligand (4-1BBL), a transmembrane molecule, member of the tumor necrosis factor ligand superfamily, is an important costimulatory molecule in the immune response. In this study a functional anti-human 4-1BBL MAb 1F1 was obtained and the specificity of this MAb was verified by flow cytometry and Western blotting. This MAb effectively recognized the 4-1BBL molecule expressed on a series of malignant cell lines as well as on DC and monocytes and it inhibited the proliferation of T lymphocytes, costimulated by soluble 4-1BBL and agonist anti-human CD3 MAb. Furthermore, we demonstrated that MAb 1F1 induced an impressive proliferation of monocytes from peripheral blood by triggering the reverse signal through 4-1BBL. This functional anti-human 4-1BBL MAb provides a valuable tool for further study of biological functions as well as signal transduction of 4-1BBL/4-1BB.     

Synthetic CD4+ T cell-targeted antigen-presenting cells elicit protective antitumor responses

CD4(+) helper T cells are critical for protective immune responses and yet suboptimally primed in response to tumors. Cell-based vaccination strategies are under evaluation in clinical trials but limited by the need to derive antigen-presenting cells (APC) from patients or compatible healthy donors. To overcome these limitations, we developed CD4(+) T cell-targeted synthetic microbead-based artificial APC (aAPC) and used them to activate CD4(+) T lymphocytes specific for a tumor-associated model antigen (Ag) directly from the naive repertoire. In vitro, aAPC specifically primed Ag-specific CD4(+) T cells that were activated to express high levels of CD44, produced mainly interleukin 2, and could differentiate into Th1-like or Th2-like cells in combination with polarizing cytokines. I.v. administration of aAPC led to Ag-specific CD4(+) T-cell activation and proliferation in secondary lymphoid organs, conferred partial protection against subcutaneous tumors, and prevented the establishment of lung metastasis. Taken together, our data support the use of cell-free, synthetic aAPC as a specific and versatile alternative to expand peptide-specific CD4(+) T cells in adoptive and active immunotherapy.     

The Wilms' tumor antigen is a novel target for human CD4+ regulatory T cells: implications for immunotherapy

Compelling evidences indicate a key role for regulatory T cells (T(reg)) on the host response to cancer. The Wilms' tumor antigen (WT1) is overexpressed in several human leukemias and thus considered as promising target for development of leukemia vaccine. However, recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T(regs). We have generated T-cell lines and clones that specifically recognized a WT1-84 (RYFKLSHLQMHSRKH) peptide in an HLA-DRB1*0402-restricted manner. Importantly, they recognized HLA-DRB1*04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a T helper 2 cytokine profile, had a CD4(+)CD25(+)Foxp3(+)GITR(+)CD127(-) T(reg) phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell contact. Priming of alloreactive T cells in the presence of T(regs) strongly inhibited the expansion of natural killer (NK), NK T, and CD8(+) T cells and had an inhibitory effect on NK/NK T cytotoxic activity but not on CD8(+) T cells. Furthermore, priming of T cells with the WT1-126 HLA-A0201-restricted peptide in the presence of T(regs) strongly inhibited the induction of anti-WT1-126 CD8(+) CTL responses as evidenced by both very low cytotoxic activity and IFN-gamma production. Moreover, these T(reg) clones specifically produced granzyme B and selectively induced apoptosis in WT1-84-pulsed autologous antigen-presenting cells but not in apoptotic-resistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 interleukin-5(+)/granzyme B(+)/Foxp3(+) CD4(+) T(regs) in five of eight HLA-DR4(+) acute myeloid leukemia patients. Collectively, our in vitro and in vivo findings strongly suggest important implications for the clinical manipulation of T(regs) in cancer patients.     

Tumor expression of 4-1BB ligand sustains tumor lytic T cells

Inadequate costimulation by solid tumors is generally believed to induce immune tolerance during primary tumor growth. We looked for tumor-specific immunity vs. tolerance in patients with Ewing's sarcoma. Circulating T cells from patients with progressively growing Ewing's tumors displayed MHC restricted tumor-induced proliferation and robust tumor lysis. Tumor-reactive T cells reside within the memory CD3+CD8+ subset and are CD28-/4-1BB+. Autologous Ewing's tumors expressed 4-1BBL, and tumor-induced T cell proliferation and activation required costimulation by 4-1BBL. Stimulation of PBL with anti-CD3/4-1BBL, but not anti-CD3/anti-CD28 induced tumor lytic effectors. Similarly, in a xenograft model, anti-CD3/4-1BBL expanded T cells controlled primary growth and prevented metastasis of autologous tumors while nonactivated and anti-CD3/anti-CD28 activated CD8+ cells did not. These results question prevailing models of tumor induced tolerance accompanying progressive tumor growth; rather, we show coexistence of progressive tumor growth and anti-tumor immunity, with costimulation provided by the tumor itself. They further demonstrate a potential new therapeutic role for 4-1BBL mediated costimulation in expanding tumor reactive CTLs for use in the adoptive immunotherapy of cancer.     

Role of immature myeloid Gr-1+ cells in the development of antitumor immunity

One of the mechanisms by which tumor cells evade the immune system is the lack of proper antigen-presenting cells. Improvement in host immunity against tumor cells can be achieved by promoting the differentiation of dendritic cells (DCs) from immature myeloid cells (Gr-1(+)Ly-6C(+)F4/80(+)) that accumulate in the bone marrow and lymphoid organs of mice with large tumor burdens. The enriched immature myeloid cells inhibit T-cell proliferation and tumor-specific T-cell response, which can be reversed by the differentiation of immature myeloid cells or depletion of F4/80(+) cells. Sorted Gr-1(+)/F4/80(+) immature myeloid cells differentiated into CD11c(+) cells that express CD80 and I-A/I-E (MHC class II) in the presence of recombinant murine granulocyte macrophage colony-stimulating factor (GM-CSF). Furthermore, intratumoral gene delivery of GM-CSF not only promoted the differentiation of carboxyfluoroscein succinimidyl ester-labeled immature myeloid cells into CD11c(+) cells with the characteristics of mature DCs (CD80(+), I-A/I-E(+)) but also enhanced innate natural killer and adaptive cytolytic T-cell activities in mice treated with interleukin (IL)-12 and anti-4-1BB combination therapy. More importantly, intratumoral delivery of GM-CSF and IL-12 genes in combination with 4-1BB costimulation greatly improved the long-term survival rate of mice bearing large tumors and eradicated the untreated existing hepatic tumor. The results suggest that inducing the maturation of immature myeloid cells, thus preventing their inhibitory activity and enhancing their antigen-presenting capability, by GM-CSF gene therapy is a critically important step in the development of effective antitumor responses in hosts with advanced tumors.     

Polarization effects of 4-1BB during CD28 costimulation in generating tumor-reactive T cells for cancer immunotherapy

Using murine tumor-draining lymph node (TDLN) cells, we investigated the polarization effect of 4-1BB (CD137) during CD28 costimulation in generating antitumor T cells. Costimulation of TDLN cells through the newly induced 4-1BB molecules, CD3, and CD28 using monoclonal antibodies significantly enhanced cell proliferation. The greater cell yield with 4-1BB signaling appeared to be related to the inhibition of activation-induced cell death. Activation of TDLN cells through 4-1BB in addition to CD3/CD28 signaling shifted T-cell responses toward a type 1 cytokine pattern because 4-1BB ligation plus CD3/CD28 stimulation significantly augmented type 1 cytokine (e.g., IFN-gamma) and granulocyte macrophage colony-stimulating factor secretion. By contrast, type 2 cytokine (e.g., interleukin 10) secretion by the activated TDLN cells was significantly reduced. The in vivo antitumor reactivity of TDLN cells activated through 4-1BB in conjunction with CD3/CD28 pathways was examined using an adoptive immunotherapy model. The number of pulmonary metastases was significantly reduced and survival was prolonged after the transfer of anti-CD3/anti-CD28/anti-4-1BB-activated TDLN cells compared with an equivalent number of cells activated without anti-4-1BB. The antitumor effect through 4-1BB involvement during CD28 costimulation was dependent on IFN-gamma production and abrogated after IFN-gamma neutralization. By contrast, interleukin 10 neutralization resulted in significantly enhanced tumor regression. These results indicate that costimulation of TDLN cells through newly induced 4-1BB and CD3/CD28 signaling can significantly increase antitumor reactivity by shifting T-cell responses toward a type 1 cytokine pattern while concomitantly decreasing type 2 responses.     

4-1BB/4-1BBL在胃癌组织及其外周血中的表达及临床意义

目的探讨胃癌局部免疫与全身免疫状态。方法应用流式细胞术,测定胃癌患者癌组织及外周血中T淋巴细胞亚群、NK细胞水平,同时测定癌组织、癌周正常胃黏膜、淋巴结及外周血单个核细胞中4-1BB、4-1BBL含量。结果胃癌患者外周血中CD3^＋、CD4^＋、CD4^＋/CD8^＋比值和NK细胞表达水平均显著高于胃癌组织（P〈0.05）,CD8^＋细胞计数反之（P〈0.05）。4-1BB表达量由低到高的顺序为外周血单个核细胞、正常胃黏膜、淋巴结、癌组织、癌组织中单个核细胞;其中癌组织明显高于正常黏膜（P〈0.05）,有统计学意义;外周血单个核细胞中4-1BB表达量明显低于其他组织,有统计学意义（P〈0.01）。癌组织中4-1BBL表达量低于正常黏膜,有统计学意义（P〈0.05）。癌组织中4-1BB表达量与CD4^＋/CD8^＋比值、NK细胞表达水平呈负相关关系,4-1BBL与CD4^＋/CD8^＋比值、NK细胞表达水平呈正相关关系,有统计学意义（P〈0.05）。结论胃癌组织内免疫力低下,处于免疫抑制状态,胃癌组织的低免疫状态与4-1BBL缺乏有关;4-1BB与胃癌患者局部和全身的免疫状态亦有密切关系。总之,胃癌组织中4-1BB/4-1BBL的表达失衡与胃癌的免疫逃逸、低免疫状态有关。     

Enhanced antitumor activity mediated by human 4‐1BB‐engineered T cells

4‐1BB (CD137) is a costimulatory molecule transiently expressed on the T‐cell surface after TCR engagement, whereas its ligand 4‐1BBL can be found on professional antigen‐presenting cells, but more importantly, also on tumor cells. As the role of the 4‐1BB/4‐1BBL pathway has emerged central to CD8⁺ T‐cell responses and survival, we sought to test its relevance in the context of genetically modified human T cells. To that end, T cells purified from healthy donors and from vaccinated‐melanoma patients were transduced to express high levels of constitutive 4‐1BB. 4‐1BB‐transduced T cells were cocultured with melanoma tumor lines and exhibited enhanced cytokine secretion, upregulation of activation markers as well as increased cytotoxicity in a chick‐chorioallantoic membrane model of human melanoma tumors. In addition, these cells expanded and proliferated at a higher rate, expressed heightened levels of the antiapoptotic molecule Bcl_XL and were also relatively insensitive to immunosuppression mediated by transforming growth factor‐β, compared to control cells. We also show that 4‐1BBL expression on the target cell is essential to 4‐1BB‐mediated functional improvement. Overall, we conclude that the modification of human T cells with 4‐1BB yields enhanced antitumor function which may have important applications in therapies based on the genetic modification of patient lymphocytes.     

PD-1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation

Tumor relapses remain a serious problem after allogeneic stem cell transplantation (alloSCT), despite the long-term persistence of minor histocompatibility antigen (MiHA)-specific memory CD8(+) T cells specific for the tumor. We hypothesized that these memory T cells may lose their function over time in transplanted patients. Here, we offer functional and mechanistic support for this hypothesis, based on immune inhibition by programmed death-1 (PD-1) expressed on MiHA-specific CD8(+) T cells and the associated role of the PD-1 ligand PD-L1 on myeloid leukemia cells, especially under inflammatory conditions. PD-L1 was highly upregulated on immature human leukemic progenitor cells, whereas costimulatory molecules such as CD80 and CD86 were not expressed. Thus, immature leukemic progenitor cells seemed to evade the immune system by inhibiting T-cell function via the PD-1/PD-L1 pathway. Blocking PD-1 signaling using human antibodies led to elevated proliferation and IFN-γ production of MiHA-specific T cells cocultured with PD-L1-expressing leukemia cells. Moreover, patients with relapsed leukemia after initial MiHA-specific T-cell responses displayed high PD-L1 expression on CD34(+) leukemia cells and increased PD-1 levels on MiHA-specific CD8(+) T cells. Importantly, blocking PD-1/PD-L1 interactions augment proliferation of MiHA-specific CD8(+) memory T cells from relapsed patients. Taken together, our findings indicate that the PD-1/PD-L pathway can be hijacked as an immune escape mechanism in hematological malignancies. Furthermore, they suggest that blocking the PD-1 immune checkpoint offers an appealing immunotherapeutic strategy following alloSCT in patients with recurrent or relapsed disease.     

CD8+ Foxp3+ regulatory T cells mediate immunosuppression in prostate cancer.

PURPOSE: Although elevated proportions of CD4(+)CD25(+) regulatory T (Treg) cells have been shown in several types of cancers, very little is known about the existence and function of CD8(+) Treg cells in prostate cancer. In this study, we investigated prostate tumor-derived CD8(+) Treg cells and their function. EXPERIMENTAL DESIGN: Tumor-infiltrating lymphocytes (TIL) from fresh tumor specimens of patients with prostate cancer were generated and subjected to phenotypic and suppressive function analyses. In particular, we investigated the role and function CD8(+) Treg cells in prostate cancer. RESULTS: We show that high percentages of CD4(+)CD25(+) T cells are probably present in the majority (70%) of prostate TILs. Remarkably, both CD4(+) and CD8(+) T-cell subpopulations possessed potent suppressive activity. T-cell cloning and fluorescence-activated cell sorting analyses showed the presence of CD8(+)CD25(+) Treg cell clones that expressed FoxP3 and suppressed na?ve T-cell proliferation, in addition to the previously known CD4(+)CD25(+) Treg cells. These CD8(+) Treg cells suppressed na?ve T-cell proliferation mainly through a cell contact-dependent mechanism. Importantly, the suppressive function of CD8(+) Treg cells could be reversed by human Toll-like receptor 8 (TLR8) signaling. CONCLUSION: Our study shows that like CD4(+)CD25(+) Treg cells, CD8(+) Foxp3(+) Treg cells present in prostate tumor-derived TILs suppress immune responses and that their suppressive function can be regulated by TLR8 ligands, raising the possibility that the manipulation of Treg cell function by TLR8 ligands could improve the efficacy of immunotherapy for prostate cancer patients.     

Peptidome from renal cell carcinoma contains antigens recognized by CD4+ T cells and shared among tumors of different histology.

PURPOSE: Renal cell carcinoma (RCC) is considered immunogenic; nonetheless, rare tumor-associated antigens have been identified or are expressed in RCC. Peptidome (i.e., the total content of natural peptides of whole cells) from other tumors, such as melanoma, has proved to be immunogenic. The aims of this study were to determine whether peptidome from RCC is immunogenic and whether it contains tumor peptides shared among allogenic RCCs. EXPERIMENTAL DESIGN: Autologous dendritic cells pulsed with RCC peptidome were used to activate in vitro CD4(+) T cells from healthy donors and a metastatic RCC patient. CD4(+) T-cell polyclonal lines and clones were characterized for tumor cell recognition by proliferation assay, killing activity, and cytokine secretion. RESULTS: CD4(+) T-cell lines and clones recognized HLA-DR-matched allogenic RCC and, for the patient, the autologous tumor. RCC-reactive CD4(+) T cells showed a heterogeneous Th1 or Th0/Th2 pattern of cytokine secretion. Moreover, RCC-reactive CD4(+) T cells recognized also melanoma, colon carcinoma, cervical carcinoma, pancreas carcinoma, lung carcinoma, gastric carcinoma, and lymphoma cells but not autologous T-cell blasts. CONCLUSIONS: Our results show that (a) the RCC peptidome contain antigens recognized by CD4(+) T cells and (b) shared among tumors of different histology and (c) it induces both Th1-type and Th2/Th0-type immune responses. These data support the use of the peptidome from allogenic RCC for specific immunotherapy in RCC and possibly in other neoplastic diseases. Moreover, the CD4(+) T-cell clones generated here are useful tools for tumor antigen identification.     

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.     

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.     

Combination therapy with cisplatin and anti-4-1BB: synergistic anticancer effects and amelioration of cisplatin-induced nephrotoxicity

Anti-4-1BB and cisplatin showed synergistic anticancer effects in the CT-26 colon carcinoma model, producing complete regression in >60% of mice with either preventive or therapeutic treatment. The tumor-free mice formed long-lasting CD8(+) T cell-dependent tumor-specific memory. Anti-4-1BB induced rapid repopulation of T and B cells from cisplatin-mediated lymphopenia and differentiation and expansion of IFN-gamma(+)CD11c(+)CD8(+) T cells. Cisplatin facilitated expansion of na?ve, effector, and memory CD8(+) T cells; combination therapy produced almost twice as many lymphoid cells as anti-4-1BB alone. Cisplatin increased 4-1BB on antigen-primed T cells and induced 4-1BB de novo on kidney tubular epithelium. Cross-linking of 4-1BB protected the T cells and kidney epithelium from cisplatin-mediated apoptosis by increasing expression of antiapoptotic molecules. Thus, cisplatin-induced 4-1BB provided a mechanism for amelioration of the lymphopenia and nephrotoxicity inherent in cisplatin treatment. We concluded that chemoimmunotherapy with anti-4-1BB and cisplatin is synergistic in tumor killing and prevention of organ-specific toxicity.     

NK and CD8+ T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.