Eradication of established renal cell carcinoma by a combination of 5-fluorouracil and anti-4-1BB monoclonal antibody in mice

Renal cell carcinoma (RCC), one of the most incurable malignancies, is highly resistant to chemotherapy and radiotherapy. Cytokine immunotherapy has been the standard approach, but the overall response rate is still very low. Administration of agonistic anti-4-1BB monoclonal antibody (mAb) has been shown to induce regression of several animal tumors but its effect on RCC is unknown. We show here that monotherapy with either anti-4-1BB mAb or the cytotoxic drug, 5-fluorouracil (5-FU), has little effect on established RCC, Renca tumors, but combination therapy with anti-4-1BB mAb and 5-FU eradicates the tumors in more than 70 % of mice. The regressing tumor tissues from mice receiving the combination therapy contained more apoptotic tumor cells and tumor infiltrating lymphocytes than tumor tissues from mice receiving 5-FU or anti-4-1BB mAb monotherapy. The number of lymphocytes in the spleens and tumor- draining lymph nodes (TDLNs) of the combination therapy mice was greatly increased compared to that of control or 5-FU monotherapy mice. Mice that had recovered due to the combination therapy rapidly rejected rechallenge with the tumor, pointing to the establishment of long-lasting tumor-specific memory. Our results indicate that targeting tumors with 5-FU, and immune cells with 4-1BB stimulation, could be a useful strategy for treating incurable RCC.     

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.     

Vaccination with dendritic cells pulsed with apoptotic tumors in combination with anti-OX40 and anti-4-1BB monoclonal antibodies induces T cell-mediated protective immunity in Her-2/neu transgenic mice

Tumor cells express tumor-associated antigens (TAAs), which can serve as targets for the immune system. However, the majority of TAAs are overexpressed products of normal cellular genes; as such, self-tolerance mechanisms have hindered their use for the induction of effective antitumor responses. One such normal self-protein is the growth factor receptor Her-2/neu, which is overexpressed in 25-35% of all mammary carcinomas in humans. In previous studies, we have demonstrated that Her-2/neu mice are functionally tolerant to neu antigens and contain only a low avidity T-cell repertoire to neu antigens. However, this residual low-avidity T-cell repertoire has antitumor activity. In this study, we compared the immune responses of Her-2/neu mice immunized with dendritic cells (DCs) pulsed with soluble neu protein or with apoptotic tumor cells. Analysis of the antitumor response shows that Her-2/neu mice vaccinated with DCs pulsed with Her-2/neu antigens retard tumor growth; however, vaccination with DCs pulsed with apoptotic tumor cells induces a stronger antitumor effect. Administration of multiple immunizations in combination with the costimulatory agonist anti-OX40 or anti-4-1BB MAb significantly enhanced the immune responses in these mice, resulting in complete tumor rejection if the tumor burden was small and substantial tumor reduction with a larger tumor burden. These results have important implications for the design of tumor vaccination strategies, suggesting that the use of vaccines that stimulate a broad immune response in combination with costimulatory molecules as immunomodulators could significantly improve the antitumor immune response in tolerant hosts.     

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c⁺CD8⁺ T cells. The CD11c⁺CD8⁺ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c⁺CD8⁺ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.     

Mantle cell lymphomas acquire increased expression of CCL4, CCL5 and 4-1BB-L implicated in cell survival

We have analyzed mantle cell lymphomas (MCLs), using high-density DNA microarrays, and confirmed the expression of differentially regulated antigens, using flow cytometry and immunohistochemistry. The results show that MCLs acquire expression of molecules that normally are involved in interaction with other immune cells and, thus, might affect the ability of the tumor to survive. The MCL signature is represented by the overexpression of the chemokine CCL4 (MIP-1beta), implicated in the recruitment of regulatory T cells, as well as CCL5 and 4-1BB-L. The latter molecules are normally involved in chemotaxis of T cells and B cell activation, respectively. Signaling through 4-1BB-L allows B cells to proliferate and the expression of its ligand, by the intra-tumoral mesh of follicular dendritic cells (FDC), could thus serve as a paracrine loop facilitating growth and survival of MCL cells.     

Suppression of human melanoma tumor growth in SCID mice by a human high molecular weight-melanoma associated antigen (HMW-MAA) specific monoclonal antibody

The lack of efficacy of available therapies for the treatment of malignant melanoma has emphasized the need to develop novel therapeutic strategies to prevent melanoma growth. We have tested whether the anti-HMW-MAA mAb 225.28S is able to inhibit human melanoma tumor growth in SCID mice because in vitro data suggested that this antigen plays a role in spreading, migration and invasion of melanoma cells. Tumors were established by subcutaneous injection of the human melanoma cell line 518A2 into SCID mice. When tumors reached a size of 5 mm, the mAb 225.28S was administered intravenously 4 times in 3 day intervals at 100 microg/injection. Within 14 days after the first administration of the mAb 225.28S, tumor growth was reduced by 52% as compared to control mice. Three hundred and seven genes of >20,000 genes contained on the GeneChip were changed in their expression level at least 2-fold after administration of the mAb 225.28S. The encoded proteins were mostly components or modifiers of the extracellular matrix, tumor suppressors, and melanogenesis associated proteins. Surprisingly, the administration of the control mAb that did not lead to a significant tumor growth inhibition in vivo resulted in the modulation of two-thirds of these genes. This is the first report of suppression of human melanoma tumor growth in SCID mice by the mAb 225.28S. Our results suggest that anti-HMW-MAA mAbs may represent useful reagents to apply passive immunotherapy to patients with malignant melanoma.     

Enhanced antitumor activity of anti-epidermal growth factor receptor monoclonal antibody IMC-C225 in combination with irinotecan (CPT-11) against human colorectal tumor xenografts.

Colon carcinomas frequently express the epidermal growth factor receptor (EGFR), and this expression correlates with more aggressive disease and poor prognosis. Previous studies have shown that EGFR blockade by monoclonal antibody IMC-C225 can inhibit the growth of human colon carcinoma tumor cells in vitro and xenografts of these tumors in athymic mice. In this report, we have studied the in vivo activity of IMC-C225 combined with the topoisomerase I inhibitor irinotecan (CPT-11) using two models of human colorectal carcinoma in nude mice. IMC-C225 was tested at a dose of 1 or 0.5 mg administered q3d. CPT-11 was administered at a dose of 100 mg/kg/week or a maximum tolerated dose of 150 mg/kg/week. Treatment with the combination of IMC-C225 (1 and 0.5 mg) and CPT-11 (100 mg/kg) significantly inhibited the growth of established DLD-1 and HT-29 tumors compared with either CPT-11 or IMC-C225 monotherapy (P < 0.05). Combination therapy with IMC-C225 (1 mg) and the MTD of CPT-11 (150 mg/kg) resulted in a regression rate of 100 and 60% of established DLD-1 and HT-29 tumors, respectively. In a refractory tumor model, combined treatment with IMC-C225 and CPT-11 significantly inhibited the growth of CPT-11 refractory DLD-1 and HT-29 tumors, whereas either agent alone did not control tumor growth. Histological examination of treated tumors showed extensive tumor necrosis, decreased tumor cell proliferation, increased tumor cell apoptosis, and a marked decrease in tumor vasculature. These results suggest that EGFR blockade by IMC-C225 combined with topoisomerase I inhibitors may be an effective therapy against chemorefractory colorectal carcinoma tumors.     

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.     

Clinical use of serum TRA-1-60 as tumor marker in patients with germ cell cancer

TRA-1-60 antigen has been related to the presence of embryonal germ cell carcinoma (EC) and carcinoma in situ. Our study further investigated the clinical efficacy of TRA-1-60 as a serum tumor marker for germ cell cancer in the testis. Three groups of patients with germ cell tumors were included: Group 1, 34 patients with disseminated disease (24 nonseminomatous germ cell tumors [NSGCT] and 10 seminomatous germ cell tumors [SGCT]); this group of patients were followed during the course of chemotherapy with measurements of TRA-1-60, HCG and AFP; Group 2, 28 patients with Stage I NSGCT (22 with embryonal carcinoma [EC]-component and 6 without EC-component, median follow-up 15 months); and Group 3, 40 patients with Stage I pure SGCT (median follow-up 15 months). Seventy-eight percent of patients with disseminated EC-positive NSGCT had increased levels of TRA-1-60 before chemotherapy. After chemotherapy, levels of TRA-1-60 had dropped significantly (p < 0.01). Levels of TRA-1-60 did not normalize in 15% of NSGCT and 30% of SGCT patients after chemotherapy. This was not associated with recurrent disease. Approximately one-third of patients with Stage I NSGCT had increased values of TRA-1-60 during follow-up without having a relapse. Contrary to earlier reports TRA-1-60 is not at present useful as a tumor marker in patients with germ cell tumors. Although detecting a few early relapses the rate of false positive elevations in the tumor marker makes it unreliable in the clinical setting. Our study did confirm that elevated levels of TRA-1-60 were present in approximately 80% of patients with disseminated EC-positive NSGCT before start of chemotherapy and chemotherapy induced a significant decrease in levels of TRA-1-60. Thus, the TRA-1-60 antigen might still prove clinically useful provided that the reliability of the assay can be increased.     

The combination therapy of α‐galactosylceramide and 5‐fluorouracil showed antitumor effect synergistically against liver tumor in mice

α‐Galactosylceramide (α‐GalCer) has been reported to be therapeutic against metastatic liver tumors in mice. However, little is known regarding the efficacy of combined chemo‐immunotherapy using α‐GalCer and anticancer drugs. In this study, we evaluated the antitumor effect of the combination therapy of α‐GalCer and 5‐fluorouracil (5‐FU) against liver tumors of MC38 colon cancer cells. The liver weights of tumor‐bearing mice treated with the combination were significantly lower than those of nontreated mice and of mice treated with 5‐FU or α‐GalCer alone. No toxic effects on the liver and renal functions were observed in any of the treatment groups. α‐GalCer treatment induced significant activation of liver NK cells in vivo, but 5‐FU treatment did not. 5‐FU treatment resulted in a significant upregulation of NKG2D activating molecules (Rae‐1 and H60) and DNAM‐1 ligands (CD112 and CD155) on MC38 cells, but α‐GalCer did not. The cytolytic activity of α‐GalCer‐activated liver mononuclear cells against 5‐FU‐treated MC38 cells was significantly higher than that against nontreated cells. The increase of the cytolytic activity induced by 5‐FU partially depended on NKG2D‐Rae‐1 or H60 signals. Depletion of NK cells significantly inhibited the antitumor efficacy of 5‐FU against MC38 liver tumors, which suggested that the antitumor effect of 5‐FU partially depended on the cytolytic activity of NK cells. These results demonstrated that the combination therapy of α‐GalCer and 5‐FU produced synergistic antitumor effects against liver tumors by increasing the expression of NK activating molecules on cancer cells. This study suggests a promising new chemo‐immunotherapy against metastatic liver cancer.     

Combination immunotherapy with interleukin‐2 surface‐modified tumor cell vaccine and programmed death receptor‐1 blockade against renal cell carcinoma

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4⁺, CD8⁺ and CD8⁺interferon‐γ⁺ but not CD4⁺Foxp3⁺ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.     

Mouse monoclonal antibodies which recognize a human (beta 1-4)galactosyl-transferase associated with tumor in body fluids.

Mouse monoclonal antibodies against human (beta 1-4)galactosyl-transferase (GalT) purified from human ovarian tumor effusion fluids were prepared and characterized. GalT purified from normal human plasma showed a single diffused band in nondenaturing polyacrylamide gel electrophoresis, but GalT purified from human ovarian tumor effusion fluids showed several oligomeric bands and a monomeric band in nondenaturing polyacrylamide gel electrophoresis. These oligomeric bands were dissociated into monomer by urea treatment and polymerized by a 2-mercaptoethanol treatment. Nine monoclonal antibodies (MAb) were prepared by immunization of purified GalT from human ovarian tumor effusion fluids and classified into three groups. Type I MAbs (MAb8611, MAb8913, and MAb8919) reacted only to the GalT monomer. Type II MAbs (MAb4880, MAb8507, and MAb8628) reacted to both the GalT monomer and the GalT polymer. Type III MAbs (MAb7907, MAb8513, and MAb8677) reacted only to the GalT polymer. These MAbs except MAb7907 could recover GalT enzyme activity from effusion fluids by immunoprecipitation. A fraction passed through MAb8513 affinity chromatography still showed reactivity to MAb8919, demonstrating that an epitope of MAb8513 resides on a minor part of GalT. A sandwich immunoassay (MAb8513-MAb8628HRP) was developed, and serum samples from ovarian cancer patients and benign ovarian patients were tested. The levels of sandwich immunoassay of serum samples from cancer were elevated significantly compared to those from benign and did not necessarily correlate to total GalT enzyme activity in serum samples. These results suggested that MAb8513 (Type III) might recognize a unique GalT associated with tumor (GAT).     

Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) in human pancreatic and ovarian tumor xenograft models

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.     

Expression of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in liver tumor lesions of rat models.

BACKGROUND AND OBJECTIVES: It has been said that amino acid transporters play an important role in supplying nutrition to cells and for cell proliferation. In this study, we examined whether LAT1 and 4F2hc are closely related to tumor growth. METHODS: Rat colon cancer cells (RCN-9) were injected into the spleen of 12 male rats (inbred F344/DuCrj). In each rat, liver samples including tumor lesions were immunostained with anti-LAT1 and anti-4F2hc antibodies. The staining area of LAT1 and 4F2hc tumor lesions was calculated by computer analysis. RESULTS: Sixty-eight tumor nodules were observed in 12 livers. Out of the 68 tumor nodules, 36 nodules (52.9%) indicated a positive staining of LAT1 and 32 (47.1%) had a negative staining of LAT1. However, the LAT1 expression was scarcely detected in non-tumor areas. In terms of the 4F2hc expression, there were 56 nodules (82.4%) with 4F2hc positive and 12 (17.6) with 4F2hc negative. In addition, the expression of 4F2hc in non-tumor areas was almost the same as the expression of 4F2hc in tumor lesions. The average tumor size of the group with LAT1 positive and 4F2hc positive (n = 31) was 0.845 +/- 0.232 mm(2), which was significantly larger than that of the group with LAT1 negative and 4F2hc negative group (n = 7) (0.090 +/- 0.028 mm(2)) or the group with LAT1 positive and 4F2hc negative (n = 5) (0.097 +/- 0.025 mm(2)), respectively (P = 0.0017, P = 0.007). CONCLUSION: LAT1 was related to tumor growth. We think that LAT1 can possibly enhance its ability to promote tumor growth in cooperation with 4F2hc.     

Programmed cell death ligand-1 (PD-L1) expression combined with CD8 tumor infiltrating lymphocytes density in non-small cell lung cancer patients

Cancer immunotherapy targeting programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway has shown promising results in treatment of non-small cell lung cancer (NSCLC) patients. T cells play a major role in tumor-associated immune response. This study aimed to investigate PD-L1 expression alone and combined with CD8 tumor infiltrating lymphocytes (TILs) density in relation to clinicopathologic parameters and survival in NSCLC patients. Immunohistochemical analysis was used to evaluate PD-L1 expression and CD8 TILs density in 55 NSCLC patients. PD-L1 immunopositivity was detected in 36 (65.5%) of NSCLC cases. PD-L1 expression was significantly related to high tumor grade (p value?=?0.038) and low CD8 TILs density (p value?=?0.004), whereas no significant relations were detected between PD-L1 expression and tumor stage (p value?=?0.121), overall survival (OS) (p value?=?0.428) and progression-free survival (PFS) (p value?=?0.439). Among PD-L1/CD8 TILs density groups, PD-L1⁺/CD8^Low group was significantly associated with high tumor grade compared to PD-L1^?/CD8^high group (pairwise p?=?0.016). PD-L1⁺/CD8^Low group was significantly related to advanced tumor stage compared to PD-L1⁺/CD8^high and PD-L1^?/CD8^Low groups (pairwise p?=?0.001 and 0.013 respectively). PD-L1^?/CD8^high group exhibited the best OS and PFS whereas PD-L1⁺/CD8^low group had the poorest OS and PFS (p value?=?0.032 and 0.001 respectively). Assessment of PD-L1 combined with CD8 TILs density, instead of PD-L1 alone, suggested important prognostic relevance in NSCLC patients.