Drug evaluation: adecatumumab, an engineered human anti-EpCAM antibody.

Micromet Inc, in collaboration with Merck Serono SA, is developing adecatumumab for the potential treatment of adenocarcinomas, including breast and prostate cancer. Adecatunmumab is a fully human antibody targeting epithelial cell adhesion molecule (EpCAM). Adecatumumab is currently undergoing phase II clinical trials in breast and prostate cancer.

Major abdominal surgery increases plasma levels of vascular endothelial growth factor: open more so than minimally invasive methods.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis that is necessary for wound healing and also promotes tumor growth. It is anticipated that plasma levels would increase after major surgery and that such elevations may facilitate tumor growth. This study's purpose was to determine plasma VEGF levels before and early after major open and minimally invasive abdominal surgery. METHODS: Colorectal resection for cancer (n = 139) or benign pathology (n = 48) and gastric bypass for morbid obesity (n = 40) were assessed. Similar numbers of open and laparoscopic patients were studied for each indication. Plasma samples were obtained preoperatively and on postoperative days (POD) 1 and 3. VEGF levels were determined via ELISA. The following statistical methods were used: Fisher exact test, unmatched Student t test, Wilcoxon's matched pairs test, and the Mann Whitney U Test with P < 0.05 considered significant. RESULTS: The mean preoperative VEGF level of the cancer patients was significantly higher than baseline level of benign colon patients. Regardless of indication or surgical method, on POD3, significantly elevated mean VEGF levels were noted for each subgroup. In addition, on POD1, open surgery patients for all 3 indications had significantly elevated VEGF levels; no POD1 differences were noted for the closed surgery patients. At each postoperative time point for each procedure and indication, the open group's VEGF levels were significantly higher than that of the matching laparoscopic group. VEGF elevations correlated with incision length for each indication. CONCLUSION: As a group colon cancer patients prior to surgery have significantly higher mean VEGF levels than patients without tumors. Also, both open and closed colorectal resection and gastric bypass are associated with significantly elevated plasma VEGF levels early after surgery. This elevation is significantly greater and occurs earlier in open surgery patients. The duration and clinical importance of this finding is uncertain but merits further study.

Drug evaluation: IGN-101--an anti-EpCAM murine antibody vaccine for cancer.

Igeneon GmbH, a wholly owned subsidiary of Aphton Corp, is developing IGN-101, an anticancer vaccine containing the EpCAM-targeting aluminum-adsorbed munrine monoclonal antibody 17-1A (edrecolomab), for the potential subcutaneous treatment of epithelial cancers such as colorectal and rectal tumors. By February 2004, Igeneon was seeking to outlicense the drug.

Perioperative immunomodulation with Flt3 kinase ligand or a whole tumor cell vaccine is associated with a reduction in lung metastasis formation after laparotomy in mice.

INTRODUCTION: Laparotomy has been associated with temporary postoperative immunosuppression and accelerated tumor growth in experimental models. In a previous murine study, a whole cell vaccine plus the adjuvant monophosphoryl-lipid A was shown to be effective in decreasing the number of lung metastases that develop after laparotomy. This study was conducted to assess the impact of the adjuvant fetal liver tyrosine kinase 3 (Flt3) ligand on perioperative tumor growth when used alone or with a tumor cell vaccine. METHODS: An intravenous tumor cell injection lung metastases model was used. Sixty female A/J mice were divided into six equal groups designated (1) anesthesia control (AC), (2) AC with Flt3 ligand (ACFlt3), (3) sham laparotomy (OP), (4) OP with Flt3 ligand (OPFlt3), (5) OP with vaccine (OPVac), and (6) OP with Flt3 ligand and vaccine (OPFlt3Vac). Groups 2, 4, and 6 received daily intraperitoneal injections of Flt3 ligand (10 microg/dose with carrier) for 5 days before and 5 days after surgery. Groups 1 and 3 received similar injections of saline on the same schedule. Groups 5 and 6 were vaccinated with irradiated whole Ta3Ha tumor cells intraperitoneally three times before and twice after surgery. Immediately after surgery, all mice were injected with 10(5) Ta3Ha tumor cells via a tail vein. After 14 days, the mice were sacrificed and their lungs and tracheas were excised en bloc. Specimens were stained and counterstained with India ink and Fekete solution, and surface metastases were counted by a blinded observer. Differences between study groups were determined by analysis of variance. The peritumoral inflammatory cell infiltrate of some Flt3 and control specimens was also assessed. RESULTS: Regarding laparotomy, Flt3 ligand (mean, 1.22 metastases), whole cell vaccine (1.12 metastases), and the combination of these two agents (0.1 metastases) were each effective in significantly decreasing the number of surface lung metastases compared with surgery alone (9.88 metastases, P < .05 for all comparisons). There were no differences between the various treatment groups in regards to number of metastases. Only the combination of Flt3 and the vaccine significantly lowered the incidence of tumors (number of mice with > or =1 tumors). Histologic analysis revealed that the Flt3-treated mice demonstrated increased numbers of antigen-presenting cells surrounding the tumors compared with controls. CONCLUSIONS: Perioperative treatment with either Flt3 ligand or a whole cell tumor vaccine significantly reduced the number of lung metastases after laparotomy. The combination of the Flt3 ligand and the vaccine also decreased the incidence of metastases and was the most effective treatment. Further studies regarding perioperative immune modulation in the setting of cancer appear warranted.

Insulin-like growth factor binding protein 3 in inflammatory bowel disease.

Epithelial cell growth regulation has been reported to be altered in inflammatory bowel disease (IBD) patients. The cell growth regulatory factor, insulin-like growth factor binding protein 3 (IGFBP-3), may be partly responsible for this phenomenon. So far, IGFBP-3 levels have been assessed as values of total protein, which is a sum of bioactive intact 43- to 45-kDa protein and its inactive proteolytic cleavage fragments. We aimed to assess the levels of intact IGFBP-3 and its cleaving protease MMP-9 in IBD. Patients with IBD and controls were included. Total plasma IGFBP-3 concentration was measured in ELISA. Western blot analysis, which distinguishes between intact and cleaved IGFBP-3, was performed in order to determine the ratio of intact to total protein; this ratio was used to calculate the concentration of intact IGFBP-3. The profile of plasma proteases was evaluated in zymography and MMP-9 levels were determined in ELISA. The concentration of intact IGFBP-3 was significantly decreased in patients with moderate to severe IBD activity compared to those in remission or controls. Of note, a dramatic depletion of intact IGFBP-3 was found in 7.4% of patients with IBD. Zymography revealed that the dominant gelatinase was the pro-form of MMP-9. However, no differences in MMP-9 levels were noted between those with active disease and controls. The level of intact IGFBP-3 is decreased in IBD patients with moderate to severe disease activity. This decrease may be linked to altered IGFBP-3 production or to increased cleavage by proteases other than MMP-9.

Significant reduction of laparotomy-associated lung metastases and subcutaneous tumors after perioperative immunomodulation with flt3 ligand in mice.

Laparotomy has been associated with increased rates of tumor establishment and metastasis formation postoperatively in animal models. The purpose of this study was to determine the impact on postoperative tumor growth of perioperative upregulation of immune function via fetal liver tyrosine kinase 3 (Flt3 ligand). Two murine studies were carried out: the first utilized a lung metastases model, and the second involved a subcutaneous tumor model. Each study included four groups: anesthesia control (AC), AC plus Flt3 ligand (ACFlt3), sham laparotomy (OP), and OP plus Flt3 ligand (OPFlt3). Flt3 ligand was administered by daily intraperitoneal injection (10 mug/dose) beginning 5 days preoperatively and continuing for 1 week postoperatively. In study 1, A/J mice were given tail vein injections of 1.5 x 10(5) TA3Ha cancer cells on the day of surgery. The mice were sacrificed 14 days after surgery, the lungs processed, and the surface metastases counted by a blinded observer. In study 2 C3H/He mice were given a dorsal subcutaneous injection of 10(4) MC-2 cancer cells on the day of surgery. The mice were sacrificed 31 days after surgery, and the injection sites were evaluated for subcutaneous tumors grossly and histologically. In study 1, the median number of surface lung metastases per mouse was 166 in the OP group and 38 in the OPFlt3 (P = .021). Mice in the AC group developed a median 50 lung metastases per animal compared with mice in the ACFlt3 group who had a median of 10 metastases per mouse (P = .001). The OP group had significantly more metastases than the AC group (P = .048). In study 2, the percentage of animals that developed tumors in the AC, OP, ACFlt3, and OPFlt3 groups was 43, 80, 0, and 20, respectively. The incidence of tumors in the OPFLt3 group and the ACFlt3 group was significantly less than their respective control groups (P < .01). The difference between the OP and AC groups was not significant (P > .05). Perioperatively administered Flt3 ligand was associated with significantly fewer lung metastases and a lower incidence of subcutaneous tumor formation after laparotomy and anesthesia alone. Perioperative immunomodulation may limit untoward surgery-related oncologic effects.

Rapid increase in serum levels of matrix metalloproteinase-9 (MMP-9) postoperatively is associated with a decrease in the amount of intracellular MMP-9.

We have previously demonstrated a significant decrease in the serum concentration of intact insulin-like growth factor-binding protein (IGFBP-3) after laparotomy. IGFBP-3, a major IGF binding protein, inhibits the growth of tumor cells via several mechanisms. Our goal was to determine, in a murine model, whether matrix metalloproteinase-9 (MMP-9), a known protease of IGFBP-3, is responsible for the postoperative decrease in serum IGFBP-3 levels. Six IGFBP-3 transgenic mice on a CD-1 background were used in this study. These mice over-express human IGFBP-3. Sham laparotomy, in the form of a midline abdominal incision, was the test procedure. General anesthesia was established using ketamine and xylazine immediately before a 30-minute sham laparotomy and before preoperative blood sampling, done via retro-orbital venipuncture, 48 hours before surgery. The animals were sacrificed and blood was drawn 24 hours postoperatively. Plasma MMP-9 activity was measured using zymography at each time point (48 hours before and 24 hours after operation). MMP-9 activity was also measured in mononuclear cell lysates at both time points. Zymography analysis demonstrated significantly higher plasma levels of MMP-9 postoperatively compared with preoperative levels (81 RU vs 40 RU; P < .05). In contrast, mononuclear cell levels of MMP-9 were significantly higher preoperatively compared with postoperative levels (37.5 RU vs. 0.75 RU, P < .05). Plasma levels of MMP-9, a known protease of IGFBP-3, are significantly elevated postoperatively. In addition, mononuclear cells that store MMP-9 are depleted of it postoperatively. This suggests that rapid MMP-9 release by mononuclear cells leads to an increase in serum levels of this protease postoperatively. Further studies will elucidate mechanisms of MMP-9-related IGFBP-3 depletion.

Immunological advantages of advanced laparoscopy.

Surgical trauma causes significant alterations in host immune function. Compared with open surgery, laparoscopic surgery is associated with reduced postoperative pain and more rapid return to normal activity. Experimental data have also shown more aggressive tumor establishment and growth rates following open surgery and laparoscopic surgery. Surgery-related immunosuppression may be partly responsible for the differences in cancer growth and outcome noted. It is clear that the choice of abdominal surgical approach has immunologic consequences. Further studies are needed to better the time course and extent of surgery-related alterations in the immune system and their clinical importance. A better understanding of the impact of surgery on the immune system may provide opportunities for pharmacologic manipulation of postoperative immune function to improve clinical results.

Insulin-like growth factor-binding protein 3 inhibits growth of experimental colocarcinoma.

BACKGROUND: We have previously shown that an important cell growth regulatory protein, insulin-like growth factor-binding protein 3 (IGFBP-3) is depleted in peripheral blood after open--but not laparoscopic--surgery. We have also demonstrated that IGFBP-3 induces apoptosis of human colon cancer cells in vitro. We report here the effect of IGFBP-3 on the growth of colonic epithelial cells in vivo. METHODS: Two tumor models were used: chemically induced carcinogenesis with azoxymethane (AOM) and inoculation of syngeneic colon cancer cells. In AOM-induced carcinogenesis, wild type (WT) and IGFBP-3 transgenic (IGFBP-3-TG) CD1 mice were injected with AOM and the number of aberrant crypt foci (ACF) in the colon studied. In the syngeneic model, BALB/c mice were inoculated with CT26 cells. The control group received saline, while the test group was administered IGFBP-3 weekly. Tumor weight was assessed 2.5 weeks after establishment. RESULTS: The number of aberrant crypt foci was significantly lower in IGFBP-3 transgenic mice (1.3 +/- 1.1) compared to WT controls (6.8 +/- 6.0) (P < .001). Further, CT26 tumors were significantly smaller in BALB/c mice that received IGFBP-3 (0.364 +/- 0.165 g) than in WT controls (0.742 +/- 0.261 g) (P < .01). CONCLUSIONS: IGFBP-3 inhibits the development of colonic tumors in experimental models and may hold promise as an adjuvant therapy for patients with neoplasms.

Infliximab: mechanism of action beyond TNF-alpha neutralization in inflammatory bowel disease.

Infliximab, a chimeric antibody to tumour necrosis factor-alpha (TNF-alpha), holds much promise for the treatment of patients with Crohn's disease. On the cellular level, infliximab affects survival and, as presented by Agnholt et al. in this issue of the journal, inhibits GM-CSF (granulocyte-macrophage colony-stimulating factor) production by intestinal T lymphocytes. Future studies will reveal whether the pro-apoptotic effect of infliximab is linked to its inhibition of endogenous GM-CSF expression in T cells. Treatment of Crohn's disease, a severe chronic intestinal disorder, may at times be challenging as it can be refractory to routine therapy. Among novel therapeutic strategies, agents that neutralize tumour necrosis factor-alpha (TNF-alpha) are of particular interest because of the crucial role of TNF-alpha in sustaining chronic mucosal inflammation. The exact mechanism of the anti-TNF action, apart from direct activity that neutralizes cytokines, is not fully understood. Cellular effects of TNF-alpha neutralizing treatment include an increased susceptibility to apoptosis of intestinal mucosal T cells. A novel pathway of anti-TNF-alpha interaction with T cells has been presented in the current issue of this journal. Agnholt et al. have found that in-vivo or in-vitro administration of infliximab, a chimeric antibody to TNF-alpha, resulted in a decreased production of GM-CSF (granulocyte-macrophage colony-stimulating factor) by T cells. Infliximab related down-regulation of TNF-alpha induced GM-CSF expression may be one of the mechanisms by which this drug increases the rate of apoptosis in T cells.

Depletion of circulating insulin-like growth factor binding protein 3 after open surgery is associated with high interleukin-6 levels.

PURPOSE: We have previously shown that plasma from open, but not laparoscopic-assisted, surgery patients has increased mitogenic activity for colon cancer cells. Decreased insulin-like growth factor binding protein 3 levels, most likely the result of an open surgery-induced proteolytic activity, may account for this finding. Plasma proteases are activated by interleukin-6. This study was designed to investigate plasma insulin-like growth factor binding protein 3 and interleukin-6 levels after major open or laparoscopic-assisted surgery. METHODS: EDTA plasma was obtained from 24 patients undergoing resection for colonic adenocarcinoma. Insulin-like growth factor binding protein 3 was detected by Western blot analysis and enzyme-linked immunosorbent assay. Interleukin-6 levels were determined by enzyme-linked immunosorbent assay. The effect of insulin-like growth factor binding protein 3 on tumor growth was tested using HCT116 cells. RESULTS: In patients undergoing open surgery, enzyme-linked immunosorbent assay revealed a significant decrease in total insulin-like growth factor binding protein 3 levels on postoperative Day 1 (915.6 +/- 378.5 ng/ml) compared with preoperative levels (1267.5 +/- 407.9 ng/ml; P < 0.001). Western blots revealed a decrease in the levels of intact insulin-like growth factor binding protein 3. In patients undergoing laparoscopic-assisted surgery, levels of total and intact insulin-like growth factor binding protein 3 before surgery (1088.9 +/- 232.5 ng/ml) and on postoperative Day 1 (1,202.3 +/- 285.6 ng/ml) were comparable with no significant changes in Western blot analysis. Interleukin-6 levels were undetectable preoperatively. On postoperative Day 1, interleukin-6 concentration was higher in open surgery group (434.8 +/- 506.6 pg/ml) compared with laparoscopic-assisted surgery group (100.9 +/- 60.2 pg/ml; P < 0.0001), and correlated significantly with a decrease in plasma insulin-like growth factor binding protein 3 after open surgery (r = 0.81; P < 0.0001). CONCLUSIONS: A significant decrease in both total and free insulin-like growth factor binding protein 3 occurs after open but not laparoscopic colectomy. There is an associated increase in the levels of interleukin-6. It remains to be proven that the interleukin-6 elevations are responsible for the low insulin-like growth factor binding protein 3 level seen after open surgery.

B cell response to tumor antigens is associated with depletion of B progenitors in murine colocarcinoma.

BACKGROUND: B cells that produce antibodies to autologous tumor antigens have been found in patients with colon cancer; the significance of this phenomenon remains unknown. Normally, the elimination of autoreactive B cells occurs in the bone marrow during their maturation. We studied the production of antibodies to syngeneic tumor antigens and the maturation of bone marrow B cells in experimental colocarcinoma model. METHODS: BALB/c mice and syngeneic CT26 colon cancer cell line were used. Reactivity of serum antibodies was tested in Western blot analysis and flow cytometry against CT26 antigens. The number of bone marrow B lineage cells was evaluated with specific antibodies and flow cytometry analysis. RESULTS: A significant decrease in the number of B cell precursors occurred in tumor-bearing mice; it normalized 3 weeks after the removal of CT26 tumors. The number of mature B cells was normal. Serum antibodies from tumor-bearing mice recognized intracellular and not surface antigens of CT26 cells. CONCLUSION: Experimental colon cancer induces B cell response to intracellular, but not surface, tumor cell antigens and restricts the B cell repertoire by depleting their precursors.

Laparoscopic-assisted cecectomy is associated with decreased formation of postoperative pulmonary metastases compared with open cecectomy in a murine model.

BACKGROUND: It was shown in a murine model that sham laparotomy is associated with a higher incidence of postoperative lung metastases when compared with results seen after carbon dioxide pneumoperitoneum. Using the same tumor model, the present study was undertaken to determine if the addition of bowel resection to the operative procedure would impact the results. METHODS: Sixty mice underwent anesthesia alone (anesthesia control [AC]), laparoscopic-assisted cecectomy (LC), or open cecectomy (OC). After surgery, all animals received tail vein injections of 105 TA3-Ha tumor cells. On postoperative day 14, the lungs and trachea were excised en bloc and processed, and surface lung metastases were counted and recorded by a blinded observer. RESULTS: The mean number of surface lung metastases in the AC, LC, and OC groups was 30.9, 76.3, and 134.5, respectively. Significantly more metastases were documented after OC (P<.001) and LC (P<.05) than after anesthesia alone. Mice in the LC group had significantly fewer lung metastases (43% less) than mice in the OC group (P<.01). CONCLUSIONS: OC was associated with significantly more lung metastases than either LC or AC. Surgery-related immune suppression or trophic tumor cell stimulation occurring after surgery may contribute to this phenomenon.

Naturally occurring antibodies to epithelial cell adhesion molecule (EpCAM).

Epithelial cell adhesion molecule (Ep-CAM) is an epithelial tumor-associated antigen that is expressed by a number of normal tissues and has been used as a target in many immunotherapy studies. The purpose of this study was to determine the incidence of serum anti-EpCAM IgG (immunoglobulin G) antibodies in colon cancer and tumor-free patients and to assess the tumor protective value of anti-EpCAM antibodies. One third of both the cancer (16/48) and the control (9/27) patients had detectable antibodies. Although the mean antibody concentration was higher in cancer patients (0.048 +/- 0.120 U) than in controls (0.038 +/- 0.064 U) the difference was not statistically significant. Western blot analysis revealed reactivity to multiple HT29 cell proteins including a 40-kDa protein (presumed to be Ep-CAM). Monoclonal anti-EpCAM 323/A3 antibody did not have a tumor-protective effect in murine Ep-CAM expressing colocarcinoma. We conclude that Ep-CAM is immunogenic not only for cancer patients, but also for tumor-free individuals.

Plasma from patients undergoing major open surgery stimulates in vitro tumor growth: Lower insulin-like growth factor binding protein 3 levels may, in part, account for this change.

BACKGROUND: Plasma from laparotomized mice has been shown to stimulate in vitro tumor growth when compared to results with preoperative plasma. This study assessed the effect of plasma from patients who underwent major open (OS) or laparoscopic surgery (LS) on in vitro tumor cell growth. METHODS: Eighty-four patients undergoing major abdominal surgery were studied (45 OS, 39 LS). Peripheral blood was collected preoperatively (PreOP) and on days 1 (POD1) and 3 (POD3) after operation. HT29 human colon cancer cells were plated with samples of the plasma. Proliferation was assessed by cell counts and the bromodeoxyuridine incorporation assay. Insulin-like growth factor binding protein 3 was detected in plasma by Western blot analysis. RESULTS: Increased mitogenic activity was noted in POD1 OS plasma when compared to PreOP OS plasma results (P <.005). This increase correlated with the length of incision (r = 0.58, P <.01). No differences were noted when the PreOP LS and POD1 LS results were compared or for any of the POD3 versus PreOP comparisons. CONCLUSIONS: Major OS is associated with alterations in plasma composition that promote HT29 tumor cell proliferation in vitro. As shown, this effect was due, at least in part, to surgery-related depletion of insulin-like growth factor binding protein 3 in peripheral blood.

Isolation of native human monoclonal autoantibodies to breast cancer.

Using a unique fusion partner cell line, MFP-2, and B-lymphocytes from breast cancer patients, we developed a set of fully human monoclonal antibodies (MAbs) that bind with high specificity and sensitivity to breast cancer cells. Immunofluorescent staining of normal tissues, primary tumors, and metastatic lymph nodes demonstrates that these antibodies are specific for breast cancer of autologous and allogeneic origin. We have also determined that many of the antibodies selected based on specific binding to breast cancer cells and tissue also bind prostate cancer cells and tissue with high specificity and sensitivity. The targets of these antibodies have been localized to the cytoplasm and membrane. Biological assays for internalization and cytotoxicity demonstrated the ability of three antibodies to rapidly internalize. Our study demonstrates that isolation of native human MAbs from the natural antibody repertoire, targeted to cancer cells, is feasible and may provide a source of tools for immunotherapy.