Treatment of implanted mammary tumors with recombinant vesicular stomatitis virus targeted to Her2/neu

Vesicular stomatitis virus (VSV) is being developed for cancer therapy. We have created a recombinant replicating VSV (rrVSV) that targeted to Her2/neu expressing breast cancer cells and expresses mouse GM-CSF. We now tested the efficacy of this rrVSV in the treatment of peritoneal tumor implants of D2F2/E2 cells, a BALB/c mouse mammary tumor cell line, which was stably transfected to express Her2/neu. Mice were treated 1 day following tumor implantation with either 2 x 10(8) infectious doses rrVSV or conditioned media (CM). All control animals developed massive peritoneal tumor with a median survival of 16 days. Nine of 10 rrVSV treated mice survived long term with no evidence of tumor. rrVSV had much less efficacy in treating implants of the parent D2F2 cells that did not express Her2/neu. The median survival was 13.5 days in mice treated with CM and 21 days in those treated with rrVSV. There was one long term survivor in the rrVSV treated group. None of the rrVSV treated animals showed evidence of viral toxicity. Three of 7 long term survivors did not develop tumor when rechallenged first with D2F2/E2 and then with D2F2 cells. Both successful therapy and resistance to rechallenge were T-cell dependent. These studies demonstrate that targeted rrVSV eliminated peritoneal implants of Her2/neu expressing tumor and elicited an anti-tumor T-cell immunologic response.     

Treatment with targeted vesicular stomatitis virus generates therapeutic multifunctional anti-tumor memory CD4 T cells

A generally applicable, easy-to-use method of focusing a patient's immune system to eradicate or prevent cancer has been elusive. We are attempting to develop a targeted virus to accomplish these aims. We previously created a recombinant replicating vesicular stomatitis virus (VSV) that preferentially infected Her2/neu expressing breast cancer cells and showed therapeutic efficacy in an implanted Balb/c mouse tumor model. The current work shows that this therapy generated therapeutic anti-tumor CD4 T cells against multiple tumor antigens. CD4 T cells transferred directly from cured donor mice could eradicate established tumors in host mice. T cells were transferred directly from donor mice and were not stimulated ex vivo. Both tumors that expressed Her2/neu and those that did not were cured by transferred T cells. Analysis of cytokines secreted by anti-tumor memory CD4 T cells displayed a multifunctional pattern with high levels of interferon-γ, interleukin (IL)-4 and IL-17. Anti-tumor memory CD4 T cells traveled to the mesenteric lymph nodes and were activated there. Treatment with targeted recombinant replicating VSV is a potent immune adjuvant that generates therapeutic, multifunctional anti-tumor memory CD4 T cells that recognize multiple tumor antigens. Immunity elicited by viral therapy is independent of host major histocompatibility complex or knowledge of tumor antigens. Virus-induced tumor immunity could have great benefit in the prevention and treatment of tumor metastases.     

Importance of tumor-specific cytotoxic CD8+ T-cells in eradication of a large subcutaneous MOPC-315 tumor following low-dose melphalan therapy

We have previously demonstrated that depletion of CD8+ T-cells by the use of a monoclonal anti-Lyt-2.2 antibody abolishes the curative effectiveness of low-dose melphalan (L-phenylalanine mustard; L-PAM) therapy for BALB/c mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor and extensive metastases (Mokyr et al., Cancer Res., 49: 4597-4606, 1989). Here we show that as a consequence of low-dose L-PAM therapy, CD8+ T-cells accumulate in the s.c. tumor nodules of MOPC-315 tumor bearers. Specifically, an 80-fold increase in the number of CD8+ T-cells was seen within 5 days after the chemotherapy. Treatment of MOPC-315 tumor bearers with low-dose L-PAM in conjunction with monoclonal anti-Thy-1.2 or anti-Lyt-2.2 antibody, in contrast to treatment with monoclonal anti-L3T4 antibody, prevented the appearance of the massive CD8+ T-cell infiltrate in the s.c. tumor nodules. Fresh CD8+ T-cells derived from s.c. MOPC-315 tumor nodules that were regressing as a consequence of low-dose L-PAM therapy exhibited a potent direct lytic activity against the MOPC-315 plasmacytoma in a short-term in vitro assay. The specificity of the lytic activity exhibited by the CD8+ T-cells was illustrated not only by the inability of the CD8+ T-cells to lyse two antigenically unrelated thymomas (the WEHI 22.1 and the EL-4) and a natural killer-sensitive lymphoma (the YAC-1), but also by their relatively weak lytic activity against an antigenically related plasmacytoma (the MOPC-104E). Thus, CD8+ T-cells that infiltrate the s.c. tumor nodules of MOPC-315 tumor bearers following low-dose L-PAM therapy most likely exploit a CTL-type lytic mechanism to eradicate at least part of the large tumor burden not eliminated by the direct antitumor effects of the drug.     

Viral infection of implanted meningeal tumors induces antitumor memory T-cells to travel to the brain and eliminate established tumors

Background

Leptomeningeal metastases occur in 2%–5% of patients with breast cancer and have an exceptionally poor prognosis. The blood–brain and blood–meningeal barriers severely inhibit successful chemotherapy. We have developed a straightforward method to induce antitumor memory T-cells using a Her2/neu targeted vesicular stomatitis virus. We sought to determine whether viral infection of meningeal tumor could attract antitumor memory T-cells to eradicate the tumors.

Methods

Meningeal implants in mice were studied using treatment trials and analyses of immune cells in the tumors.

Results

This paper demonstrates that there is a blood–meningeal barrier to bringing therapeutic memory T-cells to meningeal tumors. The barrier can be overcome by viral infection of the tumor. Viral infection of the meningeal tumors followed by memory T-cell transfer resulted in 89% cure of meningeal tumor in 2 different mouse strains. Viral infection produced increased infiltration and proliferation of transferred memory T-cells in the meningeal tumors. Following viral infection, the leukocyte infiltration in meninges and tumor shifted from predominantly macrophages to predominantly T-cells. Finally, this paper shows that successful viral therapy of peritoneal tumors generates memory CD8 T-cells that prevent establishment of tumor in the meninges of these same animals.

Conclusions

These results support the hypothesis that a virally based immunization strategy can be used to both prevent and treat meningeal metastases. The meningeal barriers to cancer therapy may be much more permeable to treatment based on cells than treatment based on drugs or molecules.     

Murine monoclonal anti-idiotypic antibody as a surrogate antigen for human Her-2/neu

The anti-idiotypic (Id) monoclonal antibody (MAb) 520C9-6b (IgG1k), raised in syngenic mice against the murine anti-Her2/neu MAb 520C9 (Ab1), functionally mimics a human Her2/neu epitope and serves as a surrogate for the protein antigen. Immunization of allogeneic C57BL/6 mice and rabbits with 520C9-6b (Ab2) induced anti-Her2/neu-specific antibodies that react with antigen-positive SKBr3 cells by ELISA and FACS analysis. The immune sera inhibited binding between Ab1 and Ab2 and vice versa (binding of Ab2 to Ab1), indicating that it was a true anti-anti-Id (Ab3) antibody. The Ab3 sera or purified Ab3 specifically lysed Her2/neu-positive SKBr3 cells, but no significant lysis was observed in antigen-negative LS174T cells in an antibody-dependent cellular cytotoxicity assay. An Id-specific cellular immune response was also demonstrated in an in vitro lymphocyte proliferation assay. Furthermore, a panel of tumor tissues and tumor cells was screened for the presence of the Her2/neu epitope by its reactivity with Ab1 and Ab3 using immunohistochemistry and FACS analysis. Identical results were obtained using either Ab1 or Ab3 (Ab1'). The data indicated that anti-Id 520C9-6b can induce Her2/neu-specific antibody in experimental animals and may serve as a potential network antigen for the treatment of patients bearing Her2/neu-positive tumors.     

Importance of Lyt 2+ T-cells in the curative effectiveness of a low dose of melphalan for mice bearing a large MOPC-315 tumor

We have previously demonstrated that the curative effectiveness of a low dose (2.5 mg/kg) of melphalan (L-phenylalanine mustard; L-PAM) for mice bearing a large s.c. (approximately 20 mm in diameter) MOPC-315 tumor and extensive metastases requires the participation of T-cell-dependent antitumor immunity in tumor eradication (S. Ben-Efraim et al., Cancer Immunol. Immunother., 15: 101-107, 1983). Here we show that the Lyt 2+ T-cells, and not the L3T4+ T-cells, participate in the cure of such tumor-bearing mice by a low dose of L-PAM. Specifically, depletion of Lyt 2+ T-cells from mice bearing a large MOPC-315 tumor by treatment with monoclonal anti-Lyt 2.2 antibody abolished the curative effectiveness of the low dose of drug. In contrast, depletion of L3T4+ T-cells from mice bearing a large MOPC-315 tumor by treatment with monoclonal anti-L3T4 antibody did not reduce significantly the curative effectiveness of the low dose of drug. Histological examination of tumor nodules on various days following low-dose L-PAM therapy revealed widespread lymphocytic infiltration by Day 5 following the chemotherapy, and this infiltration was drastically reduced when the L-PAM-treated tumor bearers were treated with either anti-Thy 1.2 or anti-Lyt 2.2 antibody but not with anti-L3T4 antibody. The antitumor immunity exhibited by Lyt 2+ T-cells derived from mice which were in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy was exploited successfully to confer systemic antitumor immunity to mice bearing a barely palpable tumor. Specifically, the adoptively transferred Lyt 2+ splenic T-cells, in conjunction with a subcurative dose of L-PAM, brought about the cure of most mice. The Lyt 2+ splenic T-cells from L-PAM-treated MOPC-315 tumor bearers were also found to be capable of exerting a direct potent lytic effect against MOPC-315 tumor cells in an antigen-specific manner. Thus, it is conceivable that the direct cytotoxic activity of Lyt 2+ T-cells for MOPC-315 tumor cells is responsible, at least in part, for the ability of the Lyt 2+ T-cells from L-PAM-treated MOPC-315 tumor bearers to bring about the eradication of the tumor burden not eradicated through the direct antitumor effects of the low dose of drug.     

Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity.

Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (IL-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact animals from large size inocula did so slowly and had a high incidence of spontaneous regression. Furthermore, mice that had rejected tumors became specifically immune to challenge with uninfected parental tumor cells. IL-7-infected FSA growing in intact mice were heavily infiltrated with host T-cells that were presumably responsible for slow growth and tumor regression, and tumor cells were in the minority. Fluorescence-activated cell sorter analysis showed that there was a 530% increase in T-cells in IL-7-infected FSA compared with control tumors. CD8+ T-cells were particularly elevated, but CD4+ lymphocytes were also increased in number, as were eosinophils and basophils. The CD4+:CD8+ ratio in IL-7-infected FSA was 1:1.7 in comparison to 1:0.6 in control tumors. Lymphocytes isolated from IL-7-producing tumors had greatly enhanced cytotoxicity towards uninfected, parental FSA cells. Killing of non-cross-reacting fibrosarcoma line was also increased but to a much lesser extent. Injection of recombinant human IL-7 directly into established FSA tumors slowed their growth and, in a significant number of instances, caused complete regression. Mice that had rejected tumor became specifically immune. The dose that was needed for this effect was, however, somewhat large: 20 micrograms twice daily for 10 days. This result contrasts with the efficacy of IL-7 gene infection in stimulating responses to the same tumor. These considerations make IL-7 a good candidate for tumor-directed cytokine gene therapy.     

Cytotoxicity of Cord Blood Derived Her2/neu-specific Cytotoxic T Lymphocytes against Human Breast Cancer in vitro and in vivo

The Her2/neu oncogene encodes a transmembrane protein with homology to the epidermal growth factor receptor. Overexpression of this gene contributes to the aggressiveness of breast cancer and poor prognosis. Therefore, Her2/neu is an ideal target molecule for generating effective cytotoxic T lymphocytes (CTLs) against breast cancers. This study reports on the generation of Her2/neu-specific CTL from umbilical cord blood mononuclear cells (UCBC) using dendritic cells primed with Her2/neu-derived peptide (KIFGSLAFL, E75) for immunostimulation. The CTLs showed specific cytotoxicity to Her2/neu high expressing MDA-453 but not toward Her2/neu low expressing MDA-231 human breast cancer cells. Similarly generated CTLs stimulated with irrelevant peptide pulsed dendritic cells did not show significant cytotoxicity towards breast cancer targets. The phenotypes of cells in culture showed high percentage of CD3+, CD4+ and CD8+T cells as determined by flow cytometry. However, the antibody mediated blocking assay demonstrated that only HLA-Class I restricted CD8+ cells are involved in the cytotoxicity. Furthermore, in vivo studies showed that treatment of SCID mice bearing MDA-453 tumor with Her2/neu-specific CTLs resulted in significant inhibition of tumor growth compared to untreated tumor bearing control mice. These results demonstrate that human umbilical cord blood mononuclear cells are a good source for generating Her2/neu-specific CTLs against human breast cancer both in vitro and in vivo.     

Effect of yttrium-90-labeled anti-carcinoembryonic antigen monoclonal antibody on the morphology and phenotype of human tumors grown as peritoneal carcinomatosis in athymic mice

Grossly visible peritoneal carcinomatosis resembling that seen in man was produced in athymic mice 7 days after intraperitoneal injection of 8 x 10(5) cells of the carcinoembryonic antigen (CEA)-producing human colon carcinoma cell line LS174T. The mice received intraperitoneal injections of 40 to 160 microCi of yttrium-90 (90Y)-labeled anti-CEA monoclonal antibody (MAb). When the mice were killed 12 days after injection, a significant inhibition of tumor growth, ranging from 40% to 95%, was observed in the treated animals when compared to the growth of tumors in the untreated animals (P less than 0.001). No mortality secondary to the therapy was seen. The bone marrow was depleted significantly at the higher doses of labeled MAb, but total recovery was observed 4 weeks after treatment. Histologically, the treated tumors showed extensive radiation effects early in the posttherapy period and massive necrosis at later times. Minute foci of viable tumor remained in the periphery. New tumor outgrowths with histologic features similar to those in the untreated controls began to appear 3 weeks after therapy. The CEA expression of the treated tumors was similar to that of the untreated controls during the early posttreatment period, diminishing progressively as the tumors became necrotic. Newly grown tumor nodules in the treated animals lacked significant CEA expression both initially and at later times. Our studies suggest that therapy with 90Y-anti-CEA MAb therapy results in selection of tumor clones lacking CEA, and that a single large dose of 90Y-MAb should be more effective than multiple fractions of smaller doses.     

Immunotherapy and whole-body hyperthermia as combined modality treatment of a subcutaneous murine sarcoma

Interleukin–2 and hyperthermia have been used individually to treat a variety of tumors in both experimental and human trials. Combined adoptive immunotherapy and hyperthermia is an exciting new line of investigation. Previous work in our laboratory has shown that combined local hyperthermia and rIL-2 therapy can significantly decrease the rate of tumor growth. In this study, we investigated the effect of combined whole-body hyperthermia (WBHT) and rIL-2 on the growth of subcutaneous MCA-105 murine tumors in C57BL/6 mice. Treatment of both microscopic (day 3) and macroscopic (day 10) tumors was evaluated. In the treatment of microscopic tumors, animals received either no treatment; rIL-2 (3 × 10⁵ IU ip tid) on days 3–7; plus WBHT(41°C for 30 min) on days 3, 5, and 7; or WBHT only on days 3, 5, and 7. In treating macroscopic tumors, animals received either no treatment; rIL-2 on days 10–14; plus WBHT on days 10, 12, and 14; or WBHT only on days 10, 12, and 14. While combined treatment and WBHT alone had no significant effect on the growth of microscopic tumors, combined IL-2 and WBHT significantly reduced the rate of tumor growth of macroscopic tumors. These results suggest that the tumor microenvironment plays a critical role in combined WBHT and rIL-2 therapy, and may be due to effects of WBHT on the tumor vasculature. © 1993 Wiley-Liss, Inc.     

Some approaches to improve the therapeutic effectiveness of adoptive chemoimmunotherapy with spleen cells from melphalan-treated BALB/c mice bearing a large MOPC-315 tumor.

Spleen cells from BALB/c mice that are in the process of eradicating a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells) were previously shown to be capable of bringing about the complete regression of a large (15 to 20 mm) s.c. MOPC-315 tumor in a substantial percentage of T-cell-deficient (athymic nude) mice that had been treated with low-dose L-PAM (adoptive chemo-immunotherapy; ACIT). Here we show that aggressive depletion of CD4+T-cells by treatment both of spleen-cell donors and of recipients with anti-L3T4 monoclonal antibody (MAb) greatly improved the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT. In the light of the apparent importance of cytotoxic T-lymphocytes (CTLs) for tumor eradication in low-dose L-PAM-treated MOPC-315-tumor bearers, it is interesting that treatment of L-PAM TuB spleen-cell donors with anti-L3T4 MAb was found to result in the generation of enhanced splenic anti-MOPC-315 cytotoxicity. Although most athymic nude mice in which the tumor had apparently completely regressed following ACIT remained tumor-free, approximately 1/3 of the mice relapsed. However, a substantial percentage of the relapsing mice were rescued by a low dose of L-PAM, which was not effective in causing tumor regression in athymic nude mice bearing a comparably large tumor if the mice had not been subjected previously to ACIT. Almost all athymic nude mice that had been "cured" of a large MOPC-315 tumor by ACIT but did not resist a subsequent MOPC-315 tumor challenge were rescued by low dose L-PAM. Thus, the therapeutic effectiveness of L-PAM TuB spleen cells in ACIT may be improved by aggressive depletion of CD4+ T-cells, suggesting that a low dose of L-PAM, which leads to the acquisition of potent splenic-tumor-eradicating immunity in BALB/c mice bearing a large MOPC-315 tumor, does not eliminate completely (or possibly not at all) the inhibitory activity of CD4+ T-cells. In addition, athymic nude mice that are not endowed with fully protective tumor-eradicating immunity following ACIT still have a substantial residual anti-tumor immune potential that can be exploited to bring about eradication of a large tumor burden following low-dose L-PAM therapy.     

Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice.

The potential for radioimmunotherapy as an adjuvant treatment for early disseminated colonic cancer was investigated in an experimental lung metastasis model. Nude mice receiving intravenous injection with a suspension of human colonic cancer cells (GW-39) developed multiple (10-100) tumor nodules throughout the lungs, and more than 50% of the animals died of extensive tumor involvement within 5-10 weeks. Groups of eight or nine animals bearing 7-day-old tumor transplants were treated with a single intravenous injection of radioiodinated agents: either 0.15 or 0.30 mCi of whole IgG of the NP-4 murine monoclonal antibody (MAb) against carcinoembryonic antigen (CEA) or 0.15 or 0.30 mCi of whole IgG of Immu-31, an anti-alpha-fetoprotein (anti-AFP) MAb. Treatment of animals with 0.15 or 0.30 mCi of 131I-labeled NP-4 IgG 7 days after injection of tumor cells resulted in survival for 23 weeks after tumor implantation in four of eight and seven of nine animals, respectively. Microscopic examination revealed that over 90% of the lung tumor colonies had no evidence of surviving cells. Animals treated with 0.30 mCi of anti-AFP, an irrelevant MAb, survived 4 weeks longer than controls. Toxicity was evident in four of the 17 animals given 0.30 mCi of NP-4 IgG (specific) or anti-AFP IgG (irrelevant) MAb. These animals died within 1-3 weeks after radioantibody injection, suggesting that death was related to the radiation dose. None of the animals given 0.15 mCi of 131I-MAb died within this period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.     

Adoptive transfer via immune T-lymphocytes of effective anti-tumor immunity against a malignant rat glioma in the brain

The aim of the present study was to develop an animal model to test the therapeutic potential of purified CD4 and CD8 T-lymphocytes against the intracerebrally implanted rat glioma cell line TZ363. Peripheral immunization of donor rats was performed by subcutaneous injection of viable TZ363 tumor cells while control animals received buffer injection. Donor splenic T-lymphocytes were prepared 14 days later and enriched by immune-bead MACS sorting. FACScan analysis revealed that of the pooled and sorted cells 91% of the tumor immune group were T-lympocytes and from the control animals 96%. The purified immune CD4/CD8 T-lymphocytes (1.2 to 5x10(7) cells) were injected intraperitoneally into 12 adult rats (three groups; each four animals), which were challenged five days later by an intracerebral injection of 5x10(4) TZ363 glioma cells. Four rats received 1.4x10(7) T-cells from control animals. While 3 of 4 animals developed a brain tumor and died in the control group, all animals, which received 5x10(7) immune T-cells survived the intracerebral tumor challenge. In the other groups survival rate depended on the amount of T-cells given. All other rats were sacrificed 32 days after intracerebral grafting. No tumor was found in these animals. Our data demonstrate that an anti-tumor T-cell response can be raised against the malignant rat glioma TZ363 and that purified CD4 and CD8 T-lymphocytes from tumor immunized donors can transfer protective immunity across the blood-brain barrier into recipient rats which are tumor challenged intracerebrally.     

Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model

Combination therapies for melanoma that target immune-regulatory networks are entering clinical practice, and more are under investigation in preclinical or clinical studies. Adenosine plays a key role in regulating melanoma progression. We investigated the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibody (mAb) in combination with either modulators of adenosine receptors (AR) activation or an inhibitor of adenosine production in a murine model of melanoma. We found that treatment with APCP, selective inhibitor of the adenosine-generating nucleotidase CD73, enhanced the activity of anti-CTLA4 mAb, by improving tumor immune response. Blockade of the adenosine A2a receptor (A2aR), which plays a critical role in the regulation of T-cell functions, significantly reduced melanoma growth. Most importantly, combination therapy including an A2aR antagonist with anti-CTLA4 mAb markedly inhibited tumor growth and enhanced anti-tumor immune responses. Targeting A3R and CTLA4 was not as effective in limiting melanoma growth as targeting A2aR. These data suggest that the efficacy of anti-CTLA4 melanoma therapy may be improved by targeting multiple mechanisms of immune suppression within tumor tissue, including CD73 or A2a receptor.     

Reduced numbers of IL-7 receptor (CD127) expressing immune cells and IL-7-signaling defects in peripheral blood from patients with breast cancer

Interleukin-7-receptor-signaling plays a pivotal role in T-cell development and maintenance of T-cell memory. We studied IL-7Ralpha (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL-7 receptor-mediated downstream effects defined by STAT5 phosphorylation (p-STAT5). Reduced numbers of IL-7Ralpha-positive cells were identified in CD4+ T-cells as well as in a CD8+ T-cell subset defined by CD8alpha/alpha homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p-STAT5 expression in the range of 0-6.4% in CD4+ T-cells and 0-4% in CD8+ T-cells. Stimulation with recombinant human IL-7 for 15 min increased p-STAT5 expression up to 36-97% in CD4+T-cells and to 26-90% in CD8+T-cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL-7 as defined by STAT5 phosphorylation, despite expression of IL-7Ralpha on T-lymphocytes. T-cells were further characterized for IL- 2 and IFN-gamma production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL-2 and IFN-gamma production combined with IL-7-signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL-7-signaling, yet intact cytokine production. Reduced numbers of IL-7Ralpha-positive cells and nonresponsiveness to IL-7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T-cells from patients with breast cancer.     

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.     

Imaging and biodistribution of Her2/neu expression in non‐small cell lung cancer xenografts with 64Cu‐labeled trastuzumab PET

Non‐small cell lung carcinomas (NSCLC) overexpress the Her2/neu gene in approximately 59% of cases. Trastuzumab, a humanized monoclonal antibody, interferes with Her2 signaling and is approved for the treatment of Her2/neu overexpressing breast cancer. However, its therapeutic use in Her2/neu overexpressing NSCLC remains obscure. The present study aimed to determine the role of ⁶⁴Cu‐labeled trastuzumab positron emission tomography (PET) for non‐invasive imaging of Her2/neu expression in NSCLC. Trastuzumab was conjugated with the bifunctional chelator 1, 4, 7, 10‐tetraazacyclododecane‐1, 4, 7, 10‐tetraacetic acid (DOTA) and radiolabeled with ⁶⁴Cu. The molecular specificity of DOTA‐trastuzumab was determined in NSCLC cell lines with Her2/neu overexpression (NCI‐H2170) and negative expression (NCI‐H520). Imaging of Her2/neu expression was performed in NCI‐H2170 tumor‐bearing mice with ⁶⁴Cu‐DOTA‐trastuzumab PET and ⁶⁴Cu‐DOTA‐IgG. In vitro studies revealed specific binding of DOTA‐trastuzumab in the Her2/neu positive NCI‐H2170 cells, while no binding was seen in the Her2/neu negative NCI‐H520 cell line. Biodistribution and PET studies revealed a significantly high accumulation of ⁶⁴Cu‐DOTA‐trastuzumab in the Her2/neu overexpressing NCI‐H2170 tumor at 24 and 48 h post‐injection (21.4 ± 1.4% and 23.2 ± 5.1% injection dose/gram (% ID/g), respectively). PET imaging of Her2/neu negative NCI‐H520 tumors showed much less uptake of ⁶⁴Cu‐DOTA‐trastuzumab (4.0% ID/g). The NCI‐H2170 tumor uptake of ⁶⁴Cu‐DOTA‐trastuzumab was significantly higher than that of ⁶⁴Cu‐DOTA‐IgG (P < 0.0001). ⁶⁴Cu‐DOTA‐trastuzumab showed a very clear image of a Her2/neu positive tumor and appeared to be effective as a PET tracer for imaging of Her2/neu gene expression in NSCLC, suggesting its potential clinical use for identifying patients that might benefit from trastuzumab‐based therapy. (Cancer Sci 2010; 101: 1045–1050)     

Adenovirus-mediated intratumoral expression of immunostimulatory proteins in combination with systemic Treg inactivation induces tumor-destructive immune responses in mouse models

Tumor-associated antigens (TAAs) include overexpressed self-antigens (for example, Her2/neu) and tumor virus antigens (for example, HPV-16 E6/E7). Although in cancer patients, TAA-specific CD4+ and CD8+ cells are often present, they are not able to control tumor growth. In recent studies, it became apparent that tumor site-located immune evasion mechanisms contribute to this phenomenon and that regulatory T cells have a major role. We tested in Her2/neu+ breast cancer and HPV-16 E6/E7+ cervical cancer mouse models, whether intratumoral expression of immunostimulatory proteins (ISPs), for example, recombinant antibodies (αCTLA-4, αCD137, αCD3), cyto/chemokines (IL-15, LIGHT, mda-7) and costimulatory ligands (CD80), through adenovirus(Ad)-mediated gene transfer would overcome resistance. In both the breast and cervical cancer model, none of the Ad.ISP vectors displayed a significant therapeutic effect when compared with an Ad vector that lacked a transgene (Ad.zero). However, the combination of Ad.ISP vectors with systemic T regulatory (Treg) depletion, using anti-CD25?mAb (breast cancer model) or low-dose cyclophosphamide (cervical cancer model) resulted in a significant delay of tumor growth in mice treated with Ad.αCTLA4. In the cervical cancer model, we also demonstrated the induction of a systemic antitumor immune response that was able to delay the growth of distant tumors. Ad.αCTLA4-mediated tumor-destructive immune responses involved NKT and CD8+ T cells. In both models no autoimmune reactions were observed. This study shows that Ad.αCTLA4 in combination with systemic Treg depletion has potentials in the immunotherapy of cancer.     

Generation of CD4+ cytotoxic T lymphocytes stimulated by immobilized anti-CD3 monoclonal antibody and interleukin-2 in cancer patients

The proliferation of autologous tumor-reactive cytotoxic T lymphocytes (CTL), induced by autologous mixed lymphocyte tumor-cell culture, was remarkably enhanced by activation with immobilized anti-CD3 monoclonal antibody (MAb) and interleukin-2 (IL-2), as compared with IL-2 alone. The activated CTL exhibited high cytotoxicity against autologous tumor cells. Cytotoxicity against autologous tumor cells was inhibited by anti-HLA-DR MAb. In negative selection with immunomagnetic beads, cytotoxicity against autologous tumor cells was inhibited by the elimination of CD4⁺ cells. The major cell-surface antigens of the activated CTL were CD3⁺, CD4⁺, CD25⁺, CD45RO⁺and CD45RA^-, suggesting helper T cells, and the activated CTL produced IL-2. It is concluded that the CTL activated by immobilized anti-CD3 MAb and IL-2 were CD4 cells that had both killer and helper functions. Our findings indicate that adoptive immunotherapy using these activated CTL would be effective in cancer patients. © 1995 Wiley-Liss, Inc.