Therapeutic effects of a synthetic peptide of C-reactive protein in pre-clinical tumor models

Previous studies have shown that multilamellar vesicles (MLV) or other carriers containing purified human C-reactive protein (CRP) have therapeutic activity in preclinical tumor models. Here we evaluated the therapeutic effects of MLV containing novel synthetic peptides, derived from the structure of CRP, on the extent of (a) established lung metastases of fibrosarcoma T241 in C57B1/6 mice, (b) survival of C57B1/6 mice bearing established liver metastases of colon carcinoma MCA-38, and (c) primary tumor growth of Renca renal carcinoma in Balb/c mice. In all cases, a single synthetic CRP peptide, RS-83277, demonstrated significant antitumor effects comparable to that seen with intact CRP. Two other synthetic CRP peptides, RS-83287 and RS-83147, showed no therapeutic activity and were comparable to control MLV containing only buffer. None of the peptides contained sequences homologous with that of the phagocyte stimulant, tuftsin. Activity of MLV-encapsulated RS-83277 was dose-dependent, and a comparable dose of the soluble peptide, given either alone or following injection of buffer-MLV, was ineffective. These results demonstrate immunotherapeutic potential for a novel synthetic peptide derived from CRP, and endogenous acute-phase protein.This work was supported in part by grant CA 49950 from the National Cancer Institute, and grant 43618 from the National American Cancer Society     

Effect of recombinant human tumor necrosis factor α on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules

Summary Incubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100–200 units/ml IL-2 was used together with 10³–10⁴ units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10 000 and 20 000 but not 40 000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110 000–220 000 units (5–10 µg) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-l-l-lysinethiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B 16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B 16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific anti-B 16 antibodies, and the addition of IL-2 further increased this anti-tumor effect. Offprint requests to: S. A. Rosenberg, Surgery Branch, National Cancer Institute, Building 10, Room 2B42, Bethesda, MD 20892 USA     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice

Summary Human peripheral blood mononuclear cells (PBM) activated with recombinant interleukin-2 (IL-2) generate potent lytic activity (LAK) against a variety of malignant cells. IL-2 alone is sufficient for LAK generation, but high concentrations are needed to generate optimal cytotoxicity. Our recent studies based on combinations of biological agents indicated that alternative activation pathways may exist. Synergy for LAK induction was investigated using IL-2 and tumor necrosis factor- (TNF). Single-cell suspensions of primary human lung carcinomas were prepared from seven established cell lines and 32 fresh tumor specimens. Not only were all cell lines sensitive to allogeneic LAK, but also all fresh tumors were sensitive to some degree to both autologous and allogeneic LAK lysis measured by a 4-h ⁵¹Cr-release assay. LAK-mediated cytotoxicity, induced with a combination of human recombinant IL-2 (Cetus, 100 U/ml) and TNF (Genentech, 500 U/ml), showed a mean fourfold increase (range 0.7–16.3) over IL-2 alone. No lytic activity was generated from PBM incubated with media or TNF alone. The sequence dependence of adding IL-2 and TNF in enhancing cytolytic activity was also studied. In vitro kinetics data revealed that the addition of TNF 2–6 h before the addition of IL-2 greatly increased LAK activity over that obtained from the simultaneous addition of the two cytokines. These results demonstrated (a) the synergy of IL-2 and TNF for generating LAK; (b) the lysis of fresh primary lung cancer cells by LAK; and (c) the sequence dependence of IL-2 and TNF for the induction of optimal LAK activity.This work was supported by NCI Grants RO2-CA45225 and CAO 9611-01, and by an award from the Prouss Foundation     

Active specific immunotherapy with vaccinia colon oncolysate enhances the immunomodulatory and antitumor effects of interleukin-2 and interferon α in a murine hepatic metastasis model

Summary The role of cytokines as primary or adjuvant antineoplastic agents has been well established. Interleukin-2 (IL-2) and the interferons have, particularly, proven to be effective antitumor agents when given alone, and seem to act synergistically on the eradication of metastases from immunogenic tumors. Active specific immunotherapy, in the form of viral oncolysates, has also shown effectiveness in cancer therapy. Bearing this in mind, we decided to combine these agents in an adjuvant triple regimen and compare their effectiveness to other treatments in terms of tumor burden and survival in a murine colon cancer hepatic metastases model. BALB/c mice were injected with CC-36, a weakly immunogenic murine colon adenocarcinoma, intrasplenically, to produce artificial liver metastases. The animals were divided into one control group and seven treatment groups receiving either vaccinia colon oncolysate (VCO), IL-2, interferon- (IFN) alone, or combinations of these agents. Half the animals were followed for survival and the other half were sacrificed at the end of the experiment for quantification of tumor burden. The blood of the sacrificed animals was utilized in a series of immunological tests in order to demonstrate the cytolytic potential of the peripheral blood lymphocytes (PBL) in each treatment group, as well as to characterize phenotypically the cells acting as effectors. The tripleadjuvant regimen group was by far the most effective treatment group, demonstrating 100% survival and a significant reduction in tumor burden when compared to other groups. Furthermore, the PBL from the animals in this group showed 69.4% lysis of the CC-36 target cells in vitro. These effector lymphocytes were characterized as ASMG1-/Lyt2.2⁺ cytolytic lymphocytes. We conclude that these lymphocytes were stimulated by the administration of VCO and further augmented by the immunomodulation of the cytokines given in the triple regimen, and that such a regimen might prove beneficial in the treatment of established hepatic metastases from weakly immunogenic tumors.Surgical Oncology Research Fellow, supported by the Julian Rickles Fellowship     

Lack of cell-cycle-specific effects of recombinant tumor necrosis factor in vivo

Several in vitro studies have demonstrated that tumor cells arrested in the G2 and M phases of the cell cycle expressed an increased sensitivity to the tumor necrosis factor (TNF). The scope of the present study was to investigate whether this cycle dependence of TNF effects also exists in vivo. The experiments were performed by using the Lewis lung carcinoma (LLC), which had been allotransplanted to nude mice. In order to induce delays of the tumor cell cycle in G2, the animals were treated with etoposide (40 mg/kg body weight i.p.) or with local radiation (15 Gy), each increasing the G2 fraction of the LLC from 10% to 35% and 50% respectively. For combination therapy with recombinant (r)TNF, the tumor was transplanted to four groups of six mice each, one of them serving as a control group the others being treated either with a G2 inductor alone, with rTNF alone, or with rTNF and a G2 inductor combined. Administration of rTNF (125 or 250 g/kg body weight i.v.) was always carried out 24 h after therapy with etoposide or radiation when the maximum of G2 accumulation had developed. The growth behavior of the treated tumors revealed that the response of the LLC to rTNF in vivo was not improved by pretreatment with a G2 inductor and, thus, obviously lacked cell-cycle specificity. It is supposed that direct interactions of TNF with the tumor cells, which are a basic requirement for cell-cycle-linked phenomena, play a minor role in the therapeutic outcome of the LLC under in vivo conditions.     

Adoptive immunotherapy of a mouse colon carcinoma with recombinant interleukin-2 alone or combined with lymphokine-activated killer cells or tumor-immune lymphocytes

Summary We have used a BALB/c colonic adenocarcinoma (C-26) to evaluate the therapeutic potential of recombinant interleukin-2 (rIL-2) at high and low dosages in combination with or without lymphokine-activated killers (LAK) or tumor-specific, immune lymphocytes in either an adjuvant spontaneous or an artificial metastasis system. Most (80%) of the mice that underwent s.c. C-26 tumor excision were shown to die of spontaneous metastasis with lung involvement by 1–4 months after excision. Postsurgical systemic treatment with low-dose rIL-2 (3 × 10⁴ U/day, i.p.) increased the survival rate to 31% as compared to 21% (not significant) in excised controls while administration of high-dose rIL-2 (8 × 10⁴ U/day) led to 53% survival (P <0.01). Both LAK cells and C-26-tumor-immune lymphocytes given during rIL-2 treatment significantly increased the effects of rIL-2 at the low but not at the high-dose, with tumor-immune effectors resulting in the highest percentage (63%) of cures. When mice bearing 3-day artificial lung metastases of C-26 cells were treated with low- or high-dose rIL-2, in combination with or without LAK or tumor-immune lymphocytes, a highly significant reduction or abrogation of the number of lung foci was observed with all treatments, including those involving or tumor-immune lymphocytes alone. Assessment of survival benefit in these mice, however, showed survival prolongation, with 20% cures achieved by low-dose rIL-2 alone and up to 65% cures by LAK in combination with low-dose rIL-2. In this system of artificial metastasis high-dose rIL-2 alone increased the survival time but failed to cure the animals, and the addition of LAK was ineffective whereas that of tumor-immune lymphocytes led to 80% cure. These results suggest that tumorimmune lymphocytes are more effective than LAK when combined with rIL-2 and that caution is necessary in extrapolating findings obtained in artificial metastasis models.     

Interleukin-1 alpha and tumor necrosis factor-alpha (TNF alpha) inhibit growth and induce TNF messenger RNA in MCF-7 human breast cancer cells.

We studied the effects of interleukin-1 alpha (IL-1) and tumor necrosis factor-alpha (TNF), alone and in combination, on MCF-7 breast cancer cells to determine whether these cytokines alter cell growth, TNF gene expression, and TNF secretion. We found that IL-1 alone and TNF alone inhibited cell growth in a dose-dependent manner. Each cytokine arrested growth in the G0/G1 phase of the cell cycle, with maximum growth inhibition at 1000 U/ml (P less than 0.05) and 100 U/ml (P less than 0.01), respectively. However, the combination of these two cytokines did not result in greater growth inhibition or a greater percentage of cells arrested in the G0/G1 phase of the cell cycle compared with each cytokine alone. We examined the effect of exogenous IL-1 and TNF on TNF gene expression by Northern blot analysis. In the absence of any cytokine, these cells do not express TNF mRNA. Exposure to IL-1 (1000 U/ml) induced TNF mRNA at 3 h; however, mRNA levels diminished thereafter to barely detectable levels by 24 h. Exposure to TNF (1000 U/ml) also induced TNF mRNA at 3 h, but in contrast to IL-1, the level of enhanced expression persisted at these levels through 72 h of exposure. Secretion of TNF by these cells is induced by exogenous TNF, but not by IL-1. IL-1 and TNF in combination do not produce greater inhibition of growth, greater amounts of TNF mRNA at 3 h, or greater secretion of TNF than that produced by TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.     

IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma

Interleukin-12 (IL-12) has been evaluated in both preclinical and clinical immunotherapy protocols as a potential therapy for melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. This study investigated the therapeutic effect of delivering a plasmid encoding IL-12 followed by electroporation on primary and secondary tumors. Three treatments of intratumoral (i.t.) plasmid injection and electroporation resulted in 80% of mice with B16.F10 melanoma tumors being tumor free for >100 days (cure). The "cured animals" were resistant to challenge with B16 cells. In a separate experiment, B16 cells were injected on the opposite flank of the treated tumor on the day of treatment. Eighty-seven percent of control mice developed a distant tumor while only 43.8% of mice receiving two or three i.t. electroporation treatments developed a distant tumor. For examination of tumor development in the lungs, mice were injected intravenously with B16.F10 cells then treated with i.m. injections of plasmid with or without electroporation. Only 37.5% of mice receiving i.m. injections and electroporation developed nodules in the lungs compared to 87.5% of mice in the no-treatment group. The results show that administration of a plasmid encoding IL-12 with electroporation has a therapeutic effect on primary tumors as well as distant tumors and metastases.     

The addition of interleukin-2 to cyclophosphamide therapy can facilitate tumor growth of B16 melanoma

The role of interleukin-2 (IL-2) on tumor growth of B16F10 melanoma cells was assessed in two sets of mice with different immune status: normal (immunocompetent) mice and immunodeficient mice. The two sets of animals were treated with cyclophosphamide (CY) or IL-2 alone or with a combined therapy of CY+IL-2. On days 6 and 10 after tumor cell injection, we evaluated the incidence of hepatic B16 melanoma metastases and the percentage of hepatic volume occupied by metastatic tissue. We observed that the CY alone (300 mg/kg, days 3 and 8 post-tumoral inoculation) significantly reduced tumor growth in all treated mice; however, CY proved more effective in normal recipients than in immunodeficient hosts. On the other hand, whereas administration of IL-2 alone (10⁵ IU daily, from day 3 to day 7) in immunocompetent mice significantly reduced tumor growth on days 6 and 10, in immunodeficient mice, no significant differences were observed in tumor growth either on the 6th or on the 10th day, in comparison to control groups. Finally, when the combined CY+IL-2 therapy was administered, an antisynergistic effect between these therapeutic agents was achieved both in normal and in immunodeficient mice. Thus, the addition of low-dose IL-2 (25×10³ IU daily, from day 4 to day 7) to high-dose CY (300 mg/kg, days 3 and 8) significantly increased tumor growth in both the early and later periods, compared to the effect of CY alone. It is concluded that exogenous IL-2 can facilitate tumor growth of B16 melanoma cells in vivo.     

Effect of combinations of cytokines and hormones on synthesis of serum amyloid A and C-reactive protein in Hep 3B cells.

We have previously shown that induction of synthesis of the two major human acute phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP), can be accomplished in the human hepatoma cell line Hep 3B, in the presence of dexamethasone, either by conditioned medium from LPS-stimulated monocytes or by the combination of IL-6 and IL-1. Neither of these cytokines alone caused significant induction of either SAA or CRP. In the present study we extended our earlier observations by evaluating the role of dexamethasone, the effect of different concentrations of IL-6 and IL-1 alpha in combination, and the possible role of TNF-alpha in regulating synthesis of SAA and CRP. Dexamethasone alone had no effect on induction of SAA or CRP. Incubation of Hep 3B cells with conditioned medium from LPS-stimulated monocytes, in the absence of dexamethasone, led to modest induction of SAA or CRP, but addition of dexamethasone potentiated this response in a dose-dependent manner. Similar results were obtained for the effect of dexamethasone on the induction of SAA by IL-6 plus IL-1 alpha. Checkerboard titration of IL-6 and IL-1 alpha revealed that increases in concentration of either cytokine led to dose-related increases in synthesis of both SAA and CRP as long as a minimal amount of the other cytokine was present. TNF-alpha alone had no significant effect on synthesis of either SAA or CRP, but the combination of IL-6 plus TNF-alpha led to substantial induction of SAA. This combination was less effective than the combination of IL-6 plus IL-1 alpha. No detectable effect of IL-6 plus TNF-alpha was observed on CRP synthesis. Both combinations of cytokines, IL-6 plus IL-1 alpha, and IL-6 plus TNF-alpha, caused increased SAA mRNA accumulation that roughly paralleled increase in synthesis. These data indicate that IL-6, IL-1 alpha, TNF-alpha, and dexamethasone in various combinations are all capable of influencing synthesis of SAA in Hep 3B cells, whereas only IL-6, IL-1 alpha, and dexamethasone can influence CRP synthesis.     

Combined effects of interferon α and interleukin 2 on the induction of a vascular leak syndrome in mice

Summary Immunotherapy with interleukin 2 (IL-2) alone or in combination with lymphokine-activated killer cells can mediate tumor regression in mice and in man. Further dose escalation of IL-2 along with lymphokine-activated killer cells has been prevented by the development of a vascular leak syndrome produced by IL-2. Because we have found that interferon (IFN-) or tumor necrosis factor (TNF-) has synergistic antitumor effects when administered together with IL-2, we have tested the vascular leakage induced by these lymphokine combinations. We used a murine model to quantify vascular leakage by measuring the extravasation of ¹²⁵I-albumin from the intravascular space as well as the wet and dry lung weights after treatment with different cytokines. Cytokines (or Hanks balanced salt solution) were administered to C57BL/6 mice and 4 h after the last injection the vascular leak was quantified. IFN- alone did not cause extravasation of radiolabel or increase in wet lung weights, though when given in combination with IL-2, significantly greater extravasation (P<0.01) as well as increase in lung water weights (P<0.05) was observed compared to the response in mice treated with IL-2 alone. IFN- in combination with IL-2 induced significant vascular leakage earlier than the response induced by IL-2 alone. For example treatment with IFN- and IL-2 induced accumulation of 14674±605 cpm in the lungs at day 1 while IL-2 alone induced 12340±251 cpm. The degree of vascular leakage was highly related to the dose of IFN- administered along with IL-2 and increased vascular leak syndrome was evident even at low doses (5000 units) of IFN-. Immunosuppression of mice by pretreatment irradiation (500 rad) markedly decreased the development of vascular leak syndrome induced by IL-2 and IFN-. Interestingly IFN- and TNF- did not induce vascular leakage in the lungs when given alone, and did not add or synergize with IL-2 in causing the syndrome. Thus the administration of IFN- in combination with IL-2 produces a dose-limiting vascular leakage that is more severe than that caused by IL-2 alone, and may be mediated, directly or indirectly by host radiosensitive cells. Abbreviations used: LAK, lymphokine-activated killer; IFN, interferon; TNF, tumor necrosis factor; IL-2, interleukin-2     

Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults

Background

DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.

Methodology/Principal Findings

We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.

Conclusions/Significance

This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00115960","term_id":"NCT00115960"}}NCT00115960 {"type":"clinical-trial","attrs":{"text":"NCT00111605","term_id":"NCT00111605"}}NCT00111605     

Combination of the c-Met Inhibitor Tivantinib and Zoledronic Acid Prevents Tumor Bone Engraftment and Inhibits Progression of Established Bone Metastases in a Breast Xenograft Model

Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by in vivo non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells in vitro. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.     

Chemo-Immunotherapy of murine solid tumors: Enhanced therapeutic effects by interleukin-2 combined with interferon α and the role of specific T cells

Summary The aim of the present study has been to assess the therapeutic efficacy of various cytokines, singly or in combination, with and without chemotherapy (cyclophosphamide, Cy), in mice carrying advanced, weakly immunogenic tumors (MCA-105 sarcoma, M109 carcinoma). Treatment of animals with i.p. growths or experimental pulmonary metastases began 8–18 days after i.p. or i.v. tumor cell inoculation respectively. None of the cytokines tested [interleukin-2 (IL-2), interferon (IFN), tumor necrosis factor (TNF) and macrophage-colony-stimulating factor (M-CSF)] nor Cy had by itself a significant curative effect. A synergistic therapeutic effect was obtained with IL-2 or IFN (but not with TNF or M-CSF) in combination with Cy. The most efficacious regimen (65%–90% cure of mice carrying i.p. tumors) was the combination of Cy+IL-2+IFN. Preliminary experiments suggested that sequential administration of these cytokines might be more beneficial than concurrent administration. Following successful immunotherapy, long-term (3–6 months) survivors showed a tumor-specific resistance to a second tumor challenge and their spleen contained an increased number of specific antitumor cytotoxic T lymphocyte precursors (5- to 20-fold, compared to control mice). In vitro and in vivo cell-depletion experiments using monoclonal antibodies revealed that T cells (primarily CD8), but not NK cells, are crucial for the therapeutic effects. This study indicates that a potent specific antitumor T cell immunity can be elicited against advanced weakly immunogenic tumors by combining chemotherapy (Cy) with IL-2 and IFN.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute