Adoptive immunotherapy for advanced cancer patients using in vitro activated cytotoxic T lymphocytes

BACKGROUND AND OBJECTIVES: We evaluated the clinical efficacy of adoptive immunotherapy using in vitro activated cytotoxic T lymphocytes (CTL) in the treatment of patients with advanced cancer. METHODS: CTL were induced with the mixed lymphocyte and tumor cell culture method, in which lymphocytes isolated from patient peripheral blood mononuclear cells were mixed with inactivated autologous tumor cells. Activated lymphocytes were administered intravenously to 11 patients once every 2 weeks for 10 weeks (i.e., 5 doses). RESULTS: Tumor reduction and decreased tumor marker were observed in 4 patients. Notably, successful CTL induction was identified in all of these patients. In patients who did not show induction of CTL response, a decreased proportion of lymphocytes, especially CD8(+) cells, and increased levels of CD14(+) cells were frequently observed. Fluorescence-activated cell sorter analysis indicated that expression of HLA class I and costimulatory factor B7-1 molecules was diminished on tumor cells. This was partly recovered with interferon-gamma, which resulted in successful induction of a CTL response. CONCLUSIONS: It was suggested that in vitro CTL induction is difficult in patients with advanced cancer. However, once the cells were induced successfully, some favorable clinical effects were seen by the adoptive transfer of such cell populations.     

Effects of photodynamic therapy with hypericin in mice bearing highly invasive solid tumors.

The tumoricidal properties of photodynamic therapy (PDT) with hypericin (HY) were evaluated in a highly metastatic adenocarcinoma (DA3Hi) and anaplastic squamous cell carcinoma (SQ2) tumors in vivo. Photosensitization of the tumor site with hypericin (HY-PDT) reduced primary tumor development and significantly prolonged the survival of tumor-bearing (TB) mice. Of these two tumors the squamous cell carcinoma emerged as more sensitive to HY-PDT compared with DA3Hi adenocarcinoma both in vitro and in vivo. HY-PDT caused extensive tumor necrosis that was followed by local, intratumoral, and systemic inflammatory reactions. Analyses of cytokine mRNA profiles reveal increases in mRNA levels of expression confined to inflammation-related cytokines both within the tumor and also systemically (measured in spleens). However, there was no evidence for any HY-PDT-induced antitumoral immune reactions. Our results suggest that PDT with hypericin can be considered as a supplementary treatment in the management of some invasive and metastatic cancers such as squamous carcinoma and similar tumors.     

Systemic therapy of experimental breast cancer metastases by mutant vesicular stomatitis virus in immune-competent mice

In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Oncolytic vesicular stomatitis virus (VSV) is a promising novel therapeutic agent for the treatment of cancer. The aim of this study was to evaluate the potential of recombinant VSV containing the M51R mutation in the matrix (M) protein gene administered intravenously as an effective and safe therapeutic agent for treating mice with experimental breast cancer metastases. Recombinant VSV(M51R)-LacZ was generated and characterized in vitro on human and murine breast cancer cells. Breast cancer metastases were established in immune-competent Balb/c mice by intravenous injection of syngeneic 4T1 cells. The vector was infused into the tumor-bearing animals via the tail vein, and productive infection of pulmonary breast cancer lesions was assessed by X-gal stainings of frozen lung sections. To evaluate potential systemic toxicity, histology of major organs and serum chemistries were analyzed. To assess effectiveness, buffer- or vector-treated tumor-bearing mice were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. We found that VSV(M51R)-LacZ efficiently replicated and lysed human breast cancer cells but was partially attenuated in 4T1 cells in vitro. We also demonstrated that its maximum tolerated dose after intravenous infusion in normal Balb/c mice was elevated by at least 100-fold over that of the parental VSV vector containing the wild-type M gene. When VSV(M51R)-LacZ was repeatedly injected intravenously into mice bearing syngeneic 4T1 tumors, the virus was able to infect multiple breast cancer lesions in the lungs without apparent toxicities, which led to significant prolongation of animal survival (P=.003). In conclusion, systemic administration of M mutant VSV is both effective and safe in the treatment of experimental breast cancer metastases in immune-competent mice, suggesting that further development of this approach may have potential for clinical application in patients.     

Adoptive cellular therapy of human breast and colorectal tumor targets using ex vivo activated memory T lymphocytes with potentiation by cis-diamminedichloroplatinum(II)

Autolymphocyte therapy (ALT) is adoptive cellular therapy of cancer using ex vivo activation of autologous peripheral blood lymphocytes (PBL). Memory T cells are the principal effector population in ALT, with in vivo activity in patients with metastatic renal cell carcinoma (RCC) and melanoma, and ex vivo cytotoxicity against autologous tumor targets. However, the noncytolytic lymphocyte portion of ex vivo-activated memory T cells (ALT cells) may also contribute as antitumor effectors. Pre-treatment of murine and human tumor cells ex vivo with chemotherapeutic agents can enhance their susceptibility to antitumor lymphocytes ex vivo and in vivo. To determine whether cis-diamminedichloroplatinum(II) (DDP) could enhance ex vivo antitumor effects of ALT cells by immuno-modulation, human breast and colorectal carcinoma target cells were derived from both primary and metastatic surgical specimens and incubated in complete medium (CM) with DDP or in CM alone (control group). Viability of each group was confirmed by trypan blue—dye exclusion test. ALT cells were prepared from autologous PBL at surgery. Primary and metastatic tumor cells from each group were used as targets for ALT cells and levels of interferon-γ (IFN-γ) release were measured as a determination of antitumor effect and recognition. Primary tumor target cells incubated in DDP showed enhanced antitumor effects and recognition by autologous ALT cells, as measured by the IFN-γ assay compared to non-DDP-treated controls. Metastatic autologous tumor target cells demonstrated less IFN-γ release than did the primary targets, although this was enhanced by pre-treating metastatic tumor targets with DDP. ALT cells demonstrated minimal IFN-γ release when incubated with allogeneic tumor targets. These data suggest that autotumor recognition of metastatic tumor targets is comparable to that of primary lesions following ex vivo pretreatment of metastatic cells with nonlethal doses of certain chemotherapeutic agents. DDP may somehow alter the physical properties of target cells, rendering them susceptible to immune-mediated attack and the combination of ALT and DDP may lead to increased therapeutic efficacy in patients with metastatic breast and colon cancer. © Wiley-Liss, Inc.     

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.     

Effect of xenogeneic immune RNA on normal human lymphocytes against human osteosarcoma cells in vitro.

New Zealand White rabbits were immunized with whole-cell suspensions of TE-85 cells (from a human osteosarcoma) maintained in tissue culture. RNA was extracted from the lymphoid tissues of the immunized animals. Normal human peripheral blood lymphocytes were pretreated with both the whole-cell immune RNA (IRNA) and the Sephadex column-eluted fractions of the whole-cell IRNA. Significant stimulation of the cytotoxic effect of the lymphocytes was observed following whole-cell IRNA pretreatment and pretreatment with peak III fractions eluted from the column. This increase in inhibition was observed whether the target cells were TE-85 (the immunizing cells), L.M. and M.Mc. (two unrelated osteosarcoma primary cell cultures), or TE-85-M-MSV cells (a cell line capable of producing a human osteosarcoma in immunosuppressed hamsters). No inhibition was observed when cells from other types of human tumors were used as target cells. The results suggested that the transferred immunity was directed against tumor-specific osteosarcoma antigens.     

Impairment of in vitro generation of cytotoxic or T suppressor lymphocytes by Friend leukemia virus infection in mice

Spleen cells collected from DBA/2 (H-2d) mice inoculated with the polycythemic variant of Friend-Leukemia Virus Complex (FLV-P) were tested for T-dependent immune functions, such as the in vitro generation of cytotoxic T lymphocytes (CTL) and of non-specific T suppressor lymphocytes (STL). CTL were generated against H-2b splenocytes, and STL were obtained following a 5-day lymphocyte culture without stimulator cells. A progressive and severe impairment of the generation of both CLT and STL was found from 2 weeks onward after infection, being almost totally abolished 3-4 weeks after virus challenge. Suppressor cells (SC) capable of inhibiting CTL generation was detected in FLV-P bearing mice. Suppressor activity was unaffected by anti-Thy 1.2 serum and complement but was removed following iron-magnet depletion or passage through nylon-wool column. Moreover complete recovery of the competence of CTL generation was attained when FLV-P infected splenocytes were passed through nylon-wool column. It is concluded that FLV-P infection depresses T-dependent cytotoxic and suppressor responses in mice, by the appearance of non-T adherent phagocytic cells, capable of impairing CTL generation in vitro.     

Combination therapy using gemcitabine and radioimmunotherapy in nude mice with small peritoneal metastases of colonic origin

INTRODUCTION: Gemcitabine has been shown to exert a radiosensitizing effect in various epithelial cancers. The aim of the present studies was to investigate whether the efficacy of radioimmunotherapy (RIT) using the (131)I-labeled anti-CEA monoclonal antibody (MAb) MN-14 could be enhanced by coadministration of gemcitabine in nude mice with small (1-3 mm) peritoneal metastases of colonic origin. MATERIALS AND METHODS: Firstly, the maximum tolerated dose (MTD) of gemcitabine was determined, when administered intraperitoneally at two different dosing schedules (0.11-3.0 mg/mouse/administration on days 0, 3, 6, and 9, or 0.022-0.60 mg/mouse/administration on days 0, 1, 2, 3, and 4). In two separate therapy studies in which these two administration regimens were applied, the efficacy of gemcitabine monotherapy was compared to that of RIT alone (125 muCi (131)I-MN-14/mouse) or RIT combined with gemcitabine. RESULTS: When administered every 3rd day for a total of 4 administrations, or daily for 5 consecutive days, the gemcitabine was considered safe at 0.33 mg/mouse/administration and 0.066 mg/mouse/administration, respectively. In the first therapy study, median survival of the control mice was 39 days. Gemcitabine monotherapy at 0.11 mg or 0.33 mg/mouse/administration every 3rd day (total, 4 administrations) resulted in a median survival of 52 and 57 days, respectively (p = 0.0003, compared to controls). RIT alone resulted in a median survival of 66 days (p < 0.0001, compared to controls). The combination of RIT and gemcitabine coadministration resulted in a median survival of 73 and 94 days, respectively (p = 0.12, for trend). In the second therapy study, median survival of the control mice was 48 days, which was similar to the median survival of the mice treated with daily administrations of gemcitabine monotherapy at 0.022 mg/mouse/administration on 5 consecutive days (49 days; p = 0.17). RIT alone resulted in a significantly improved median survival of 66 days (p= = 0.0010, compared to controls). Combination therapy using RIT and gemcitabine resulted in a median survival of 64 days, which did not differ significantly from the survival of the mice treated with RIT alone (p = 0.43). CONCLUSIONS: At the dose regimens employed, gemcitabine did not enhance the efficacy of RIT of experimental small-volume peritoneal carcinomatosis of colonic origin.     

Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.     

Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells

Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non- or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.     

Sensitized T lymphocytes render DBA/2 beige mice responsive to IFN α/β therapy of friend erythroleukemia visceral metastases

Interferon α/β (IFN α/β) is highly effective in inhibiting the development of Friend erythroleukemia cell (FLC) visceral metastases in DBA/2 mice injected intravenously (i.v.) with FLC, but does not protect FLC-injected DBA/2 beige (bg/bg) mice. Use of IFN α/β-resistant FLC indicated that IFN was acting through host mechanisms in DBA/2 mice and thus pointed to a defect in some host mechanism in bg/bg mice essential for IFN's anti-metastatic action. We undertook experiments to restore in bg/bg mice the marked anti-FLC meta-static effect of IFN a/p observed in DBA/2 and +/bg mice. Adoptive transfer of spleen cells from normal syngeneic mice to IFN-treated bg/bg mice was ineffective, but the transfer of splenic T lymphocytes from FLC-immunized DBA/2 or +/bg mice markedly increased the survival time of FLC-injected bg/bg mice provided that these mice were also treated with IFN a/p. Neither treatment alone resulted in an increase in survival time. As few as 1 × 10⁷ immune spleen cells were effective in IFN-treated FLC-injected bg/bg mice. The T-cell immune response to FLC of bg/bg mice was diminished compared with that of +/bg mice. Likewise, only combination therapy of immune spleen cells and IFN α/β resulted in an increased survival time of ESb-lymphoma-injected bg/bg mice. Our results indicate the essential participation both of T-cell-mediated immune mechanisms and of IFN α/βin the inhibition of FLC visceral metastases.     

Anti-cancer efficacy of peplomycin, adsorbed on activated carbon particles, against lymph node metastases in mice

PEP-CH is comprised of activated carbon particles that adsorb peplomycin. Mice which were inoculated with 5 x 10(5) MH 134 tumor cells subcutaneously opposite the foot-pad of the left hind paw on day 0, received a subcutaneous injection of peplomycin, 0.25 mg/mouse, in the form of PEP-CH or a peplomycin aqueous solution administered at the foot-pad of the left hind paw on day 10. The left popliteal lymph nodes then were transferred intraperitoneally to normal mice on day 14. In the PEP-CH group, the survival time of the recipients was statistically and significantly long, and the transferred tumor cell number, estimated with a calibration curve, was markedly small when compare to mice in the other treatment groups.     

Interleukin-12 enhances the antitumor activity of cytotoxic T lymphocytes against lung adenocarcinoma engrafted in severe combined immunodeficient mice

Background. Through a number of biologic activities, interleukin 12 (IL-12) has proven to be a potential antitumor cytokine in mice bearing a variety of malignancies. However, in clinical trials in humans, the eradication of solid tumors remains difficult. Methods. A lung cancer cell line (PC-9)-specific cytotoxic T lymphocytes (CTL) were generated by multiple stimulations, with irradiated PC-9 cells, of regional lymph node lymphocytes obtained from patients with lung cancer whose cells expressed the same HLA-A locus haplotype as PC-9 (HLA-A24). Severe combined immunodeficient (SCID) mice bearing a subcutaneous graft of PC-9 were then intravenously injected with anti-PC-9-specific CTLs. Under these conditions, the in-vivo effect of recombinant human (rh) IL-2 and rh IL-12 was evaluated, based on tumor growth. Results. Mice that received either rh IL-2 or rh IL-12 exhibited no inhibitory effect on tumor growth. However, mice that received adoptive immunotherapy (AIT) alone exhibited a significant inhibition of tumor growth in the PC-9 graft in comparison to untreated mice. When mice were treated with AIT combined with rh IL-2 + rh IL-12 administration, tumor growth was significantly suppressed. A significant difference was observed in the growth of the PC-9 graft between AIT + IL-2 + IL-12 treatment and AIT + IL-2 treatment. Four of eight mice in the AIT + IL-2 + IL-12-treated group showed complete tumor regression. Conclusion. IL-12 showed a synergistic effect with adoptive immunotherapy, using CTL in a tumor-engrafted SCID model. These results are therefore considered to provide a sufficient rationale for IL-2 + IL-12-based immunotherapy using CTL transfer for patients with lung cancer. Received: June 30, 1999 / Accepted: May 10, 2000     

Depletion of alloreactive T-cells in vitro using the proteasome inhibitor bortezomib preserves the immune response against pathogens

Current graft-versus-host disease (GVHD) inhibition approaches lead to abrogation of pathogen-specific T-cell responses. We propose an approach to inhibit GVHD without hampering immunity against pathogens: in vitro depletion of alloreactive T cells with the preoteasome inhibitor bortezomib. We show that PBMCs stimulated with allogeneic cells and treated with bortezomib greatly reduce their ability to produce IFN-γ when re-stimulated with the same allogeneic cells, but mainly preserve their ability to respond to citomegalovirus stimulation. Unlike in vivo administration of immunosuppressive drugs or other strategies of allodepletion, in vitro allodepletion with bortezomib maintains pathogen-specific T cells, representing a promising alternative for GVHD prophylaxis.     

Efficient induction of cytotoxic T lymphocytes specific to hepatocellular carcinoma using HLA-A2-restricted MAGE-n peptide in vitro

MAGE-n is a new member of MAGE gene family and has been demonstrated closely associated with hepatocellular carcinoma (HCC). In this study, MAGE-n-derived peptide-specific cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with HLA-A2-restricted MAGE-n peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis against T2 cells pulsed with the peptide and HLA-A2+ HCC cells expressing MAGE-n, while HLA-A2+ HCC cell lines that did not express MAGE-n could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-MHC class I monoclonal antibody. These results suggested the MAGE-n peptide could be a potential target of specific immunotherapy for HLA-A2 patients with HCC.     

Ovarian malignant ascites-derived lymphocytes stimulated with prothymosin α or its immunoactive decapeptide lyse autologous tumour cells in vitro and retard tumour growth in SCID mice

BackgroundTumour-associated lymphocytes (TALs) present in effusions of ovarian cancer patients exhibit impaired activities, due to the immunosuppressive environment of the ascites. Means to enhance their cytotoxicity against autologous tumour cells are of clinical importance. The immunomodulator prothymosin alpha (proTα) increases the specific lysis of tumour cells by activated CD8⁺ T-lymphocytes and its immunoreactivity is exerted by the carboxy-terminal decapeptide, proTα(100-109). These two molecules were studied on TALs in vitro, and in SCID mice bearing human ovarian tumours.MethodsTALs and tumour cells were isolated from 41 ovarian cancer patients and co-cultured in the presence of proTα or proTα(100-109). The cytotoxicity of peptide-stimulated TALs was tested against autologous tumour cells and K562. Ex vivo peptide-stimulated TALs from three patients were adoptively transferred intraperitoneally in SCID mice, previously inoculated with each patient’s autologous tumour cells.ResultsProTα and its immunoreactive peptide proTα(100-109), enhanced the cytotoxic activity of TALs against autologous tumour cells in vitro, but marginally affected the lysis of K562. The effect of proTα and proTα(100-109) was higher after 7–14 days of stimulation, whereas TAL cytotoxicity was significantly decreased after 21 days. Mice administered TALs, ex vivo activated with proTα or proTα(100-109) for 7 days, showed a relatively lower tumour increase rate and a prolongation of their survial, compared to controls.ConclusionOur data demonstrate that, in the presence of tumour antigens, proTα and proTα(100-109) enhance the depressed cytotoxicity of TALs against autologous tumour cells in vitro and retard tumour growth in vivo.     

Transfer of in vitro expanded T lymphocytes after activation with dendritomas prolonged survival of mice challenged with EL4 tumor cells

Adoptive T cell transfer after in vitro expansion represents an attractive cancer immunotherapy. The majority of studies so far have been focusing on the expansion of tumor infiltrated lymphocytes (TIL) and some have shown very encouraging results. Recently, we have developed a unique tumor immune response activator, dendritomas, by fusion of dendritic cells and tumor cells. Animal studies and early clinical trials have shown that dendritomas are able to activate tumor specific immune responses. In this study, we hypothesized that na?ve T cells can be primed with dendritomas and expanded in vitro to develop an adoptive transfer therapy for patients who do not have solid tumors, such as leukemia. T cells were isolated and purified from lymph nodes of mice. The cells were then incubated with dendritomas made from syngeneic DCs and tumor cells and expanded in vitro using Dynabeads mouse CD3/CD28 T cell expander for approximately three weeks. The in vitro primed and expanded T cells showed tumor cell specific CTL activity and increased secretion of IFN-gamma. Tumor bearing mice receiving the in vitro expanded T cells survived significantly longer than control mice. Furthermore, the depletion of regulator T cells enhanced the survival of the mice that received the adoptive transfer therapy.