Increased neutrophil-lymphocyte ratio is a poor prognostic factor in patients with primary operable and inoperable pancreatic cancer.

BACKGROUND: The neutrophil-lymphocyte ratio (NLR) has been proposed as an indicator of systemic inflammatory response. Previous findings from small-scale studies revealed conflicting results about its independent prognostic significance with regard to different clinical end points in pancreatic cancer (PC) patients. Therefore, the aim of our study was the external validation of the prognostic significance of NLR in a large cohort of PC patients. METHODS: Data from 371 consecutive PC patients, treated between 2004 and 2010 at a single centre, were evaluated retrospectively. The whole cohort was stratified into two groups according to the treatment modality. Group 1 comprised 261 patients with inoperable PC at diagnosis and group 2 comprised 110 patients with surgically resected PC. Cancer-specific survival (CSS) was assessed using the Kaplan-Meier method. To evaluate the independent prognostic significance of the NLR, the modified Glasgow prognostic score (mGPS) and the platelet-lymphocyte ratio univariate and multivariate Cox regression models were applied. RESULTS: Multivariate analysis identified increased NLR as an independent prognostic factor for inoperable PC patients (hazard ratio (HR)=2.53, confidence interval (CI)=1.64-3.91, P<0.001) and surgically resected PC patients (HR=1.61, CI=1.02-2.53, P=0.039). In inoperable PC patients, the mGPS was associated with poor CSS only in univariate analysis (HR=1.44, CI=1.04-1.98). CONCLUSION: Risk prediction for cancer-related end points using NLR does add independent prognostic information to other well-established prognostic factors in patients with PC, regardless of the undergoing therapeutic modality. Thus, the NLR should be considered for future individual risk assessment in patients with PC.

Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury.

Despite substantial progress, mortality and morbidity of the acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI), remain unacceptably high. There is no effective treatment for ARDS/ALI. The renin-angiotensin system (RAS) through Angiotensin-converting enzyme (ACE)-generated Angiotensin II contributes to lung injury. ACE2, a recently discovered ACE homologue, acts as a negative regulator of the RAS and counterbalances the function of ACE. We hypothesized that ACE2 prevents Bleomycin (BLM)-induced lung injury. Fourteen to 16-week-old ACE2 knockout mice-male (ACE2(-/y)) and female (ACE2(-/-))-and age-matched wild-type (WT) male mice received intratracheal BLM (1.5U/kg). Male ACE2(-/y) BLM injured mice exhibited poorer exercise capacity, worse lung function and exacerbated lung fibrosis and collagen deposition compared with WT. These changes were associated with increased expression of the profibrotic genes α-smooth muscle actin (α-SMA) and Transforming Growth Factor ß1. Compared with ACE2(-/y) exposed to BLM, ACE2(-/-) exhibited better lung function and architecture and decreased collagen deposition. Treatment with intraperitoneal recombinant human (rh) ACE2 (2 mg/kg) for 21 days improved survival, exercise capacity, and lung function and decreased lung inflammation and fibrosis in male BLM-WT mice. Female BLM WT mice had mild fibrosis and displayed a possible compensatory upregulation of the AT2 receptor. We conclude that ACE2 gene deletion worsens BLM-induced lung injury and more so in males than females. Conversely, ACE2 protects against BLM-induced fibrosis. rhACE2 may have therapeutic potential to attenuate respiratory morbidity in ALI/ARDS.

Peptide arrays for the determination of humoral responses induced by active immunization with a monoclonal antibody against EpCAM.

Epithelial cell adhesion molecule (EpCAM) is an attractive target for monoclonal antibody serotherapy because it is over-expressed in approximately 70% of epithelial cancers and their metastatic lesions. IGN101, the immunogenic formulation of the murine monoclonal anti-EpCAM antibody Mab17-1A, has been shown to evoke a strong humoral immune response in both monkey studies and early clinical trials. Notably, there was a reduction in the number of circulating EpCAM-positive tumor cells in the peripheral blood of treated cancer patients. In contrast to earlier publications by other groups, we could not detect an anti-EpCAM immune response upon treatment with Mab17-1A using a conventional but optimized anti-EpCAM ELISA. Therefore, in a novel experimental setup, sera of healthy immunized monkeys, normal human donors and cancer patients immunized with IGN101 were tested for reactivity against a series of overlapping synthetic peptides encompassing the entire sequence of EpCAM prepared by SPOT synthesis on cellular supports. Using this method, sera from normal donors reacted with different peptides compared to sera from healthy monkeys. However, the peptides were clustered in the same regions of EpCAM. Cancer patients generally had a lower reactivity to EpCAM peptides and immunization with IGN101 induced reactivity against a different set of peptides. Antibodies cross-reacting with both the IgG2a framework and with the Mab17-1A idiotype were identified. In summary, our data indicate that some EpCAM peptides may be recognized in a species-specific manner. At least seven EpCAM-derived peptides could be of diagnostic interest (QCQCTSVGAQ, ERVRTYWIII, ALQKEITTRY, TYWIIIELKH, IADVAYYFEK, AYYFEKDVKG, GQTLIYYVDE), while four out of these seven peptides may also possess therapeutic relevance (TYWIIIELKH, ALQKEITTRY, IADVAYYFEK, AYYFEKDVKG).

Increased expression of the blood group-related Lewis Y antigen on synovial fluid granulocytes of patients with arthritic joint diseases.

OBJECTIVE: To evaluate the expression of the carbohydrate structures Lewis Y (LeY), sialyl-LeX (sLeX) and Lewis X (LeX) on paired peripheral blood (PB) and synovial fluid (SF) granulocytes in patients with arthritic diseases. METHODS: Ten patients with rheumatoid arthritis (RA), seven patients with spondyloarthritis (SA) and eight patients with osteoarthritis (OA) were studied. Granulocyte expression of the Le oligosaccharides was analysed by fluorescence-activated cell sorting. RESULTS: SF granulocytes of patients with RA, SA and OA expressed higher levels of the LeY oligosaccharide than PB granulocytes. Increases in LeY on SF granulocytes were similar in all three underlying diseases. No differences in the expression of the Le antigens were detected between PB granulocytes of patients and healthy individuals. Expression of sLeX and LeX showed no variation between SF and PB neutrophils. CONCLUSION: The selective increase in LeY antigen on SF granulocytes in RA, SA and OA suggests a role of the LeY oligosaccharide in granulocyte traffic and inflammatory responses.

Activation-dependent expression of the blood group-related lewis Y antigen on peripheral blood granulocytes.

The expression of the difucosyl-lactosamine type 2 oligosaccharide Lewis Y (LeY) on peripheral blood cells was investigated. As assessed by the reactivity with the mouse anti-LeY monoclonal antibody (mAb) ABL 364 among circulating blood cells, the expression of the LeY oligosaccharide was uniquely restricted to granulocytes. Although the density of LeY expressed on resting granulocytes was weak, in vitro activation of granulocytes with fMLP induced a rapid and pronounced increase in granulocyte LeY expression. Analysis of CEA-related glycoproteins immunoprecipitated with anti-CD66 mAbs followed by immunoblotting with mAb ABL 364 showed that granulocyte LeY is attached to members of the CD66 cluster, in particular to the 160/90 kD glycoprotein recognized by anti-CD66 mAb CBL/gran 10. The activation-associated increase in LeY attached to CD66 adhesion molecules implicates a role of the LeY determinant in the cytoadhesive properties of granulocytes.

Different types of FCgamma-receptors are involved in anti-Lewis Y antibody induced effector functions in vitro.

Stimulation of monocytes by interaction of monoclonal antibodies (mAbs) with Fc gamma receptors (FcgammaRs) results in the activation of various monocyte effector functions. In the present investigation we show that the anti-Lewis Y (LeY) anti-tumour mAb ABL 364 and its mouse/human IgG1 chimaera induce both antibody-dependent cellular cytotoxicity (ADCC) and the release of tumour necrosis factor alpha (TNF-alpha) during mixed culture of monocytes with LeY+ SKBR5 breast cancer cells in vitro. Although anti-LeY mAb-mediated TNF-alpha release paralleled ADCC activity, cytokine release required a higher concentration of sensitizing mAb than the induction of cytolysis. The determination of the FcgammaR classes involved in the induction of the distinct effector functions showed that anti-LeY mAb-induced cytolysis was triggered by interaction between anti-LeY mAbs and FcgammaRI. In contrast, mAb-induced TNF-alpha release mainly depended on the activation of monocyte FcgammaRII. Neutralization of TNF-alpha showed no influence on monocyte ADCC activity towards SKBR5 target cells. Our data indicate an independent regulation of anti-LeY mAb induced effector functions of ADCC and TNF-alpha release which seemed to be triggered by activation of different types of FcgammaR.

A double-blind randomized-phase II trial comparing immunization with antiidiotype goat antibody vaccine SCV 106 versus unspecific goat antibodies in patients with metastatic colorectal cancer.

This article reports on the first double-blind randomized clinical study with an antiidiotype antibody vaccine in patients with metastatic colorectal carcinoma. The study was performed to determine immunological parameters, efficacy, and tolerability of the vaccine. Forty-two patients with metastatic colorectal cancer were randomly assigned to multiple immunizations with goat IgG antiidiotype vaccine SCV 106 (n = 21) or unspecific goat IgG as controls (n = 21). The antiidiotype vaccine mimicked the 17-1A glycoprotein antigen associated with colorectal cancer. Of the 42 patients entered, 39 were evaluable for efficacy (SCV 106, n = 18; controls, n = 21). Twenty-nine patients raised antibodies to the vaccines (immunological responders, SCV 106, n = 12; controls, n = 17). Only in the SCV 106 group was a significant increase (p = 0.002) of titers with specificity of antitumor antibody 17-1A found. According to the International Union Against Cancer (UICC) criteria no tumor response was observed. However, in the SCV 106 group the relative increase of carcinoembryonic antigen (CEA) levels between entry and observed disease progression was lower (p = 0.03) and disease progression was determined less frequently by development of new metastases (p = 0.001). On an intention-to-treat basis, the survival time difference between the two groups was not significant. Comparison of immunological responders in both groups revealed a significant survival advantage of the SCV 106-treated patients compared with controls (mean 67 versus 39 weeks; p = 0.01). Immunizations were well tolerated. Vaccination of immunologically responding metastatic colorectal carcinoma patients with SCV 106 leads to slowed disease progression and tumor dissemination and significantly prolongs survival time.

Humanized anti-Lewis Y antibodies: in vitro properties and pharmacokinetics in rhesus monkeys.

ABL 364 is a murine monoclonal IgG3 antibody directed against the Lewis Y carbohydrate antigen (Le(y)) expressed on the surface of many epithelial cell tumors. The antibody mediates cytotoxicity via activation of human complement or human effector cells, and has been evaluated in several clinical trials including two Phase I/II trials in relapsed small cell lung cancer and metastatic breast cancer. To improve the effector functions of the antibody, increase its half-life in circulation, and avoid the human antimouse antibody response, two chimeric and several humanized antibodies were constructed for evaluation. The chimeric IgG1 is more potent than the murine IgG3 in tumor cell lysis via activation of human peripheral mononuclear cells (10-fold), but somewhat less effective in complement-dependent lysis (2-3 fold). The chimeric IgG3 is slightly less potent than the IgG1. A humanized IgG1 was constructed by combining the complementarity-determining regions of the ABL 364 antibody with human framework and constant regions. Several additional variants were subsequently constructed to improve the binding affinity and increase expression of the antibody. Two of the variants, designated I and K, differ by a single amino acid at position 75 of the heavy chain. Both variants have affinity within 2-fold of the chimeric IgG1 antibody and retain the cytolytic activities toward tumor cell lines. However, it was possible to express variant K at a significantly higher level (5- 10-fold) than variant I. Pharmacokinetics of the humanized ABL 364 antibody variant K was compared with that of the parent murine antibody in rhesus monkeys. It was shown that the terminal half-life of the humanized antibody in rhesus monkeys is 14-20 days, with a mean of 16.3 days, while that of the parent murine antibody is only 1.9 days.

Reduction of metastatic carcinoma cells in bone marrow by intravenously administered monoclonal antibody: towards a novel surrogate test to monitor adjuvant therapies of solid tumours.

In a pilot, prospective randomised study, 40 patients with breast and colorectal cancer presenting with metastatic cytokeratin (CK)-positive tumour cells in bone marrow were treated either with six doses of 100 mg of a monoclonal Lewis Y antibody during 2 weeks or with a placebo regimen, consisting of six infusions of human serum albumin (HSA). CK-positive cells in marrow were monitored prior to and on days 15 and 60 after commencement of treatment. In 30 patients presenting with relatively low tumour cell numbers (1-11 per 4 x 10(5) bone marrow cells), a therapy-induced reduction of CK-positive cells could not be conclusively determined. More meaningful quantitative data were obtained in 10 breast cancer patients presenting with more than 20 tumour cells per 4 x 10(5) nucleated bone marrow cells. 7 of these patients had been randomised to the antibody arm, and 5 showed an eradication or a distinct reduction of CK-positive/Lewis Y-positive cells of at least one log unit, while 2 patients, presenting with Lewis Y-negative tumour cells, showed no corresponding decrease. Similarly, in all 3 patients randomised to the placebo arm, tumour cells were not reduced. Because the antibody exerted a marked cytotoxicity on tumour cell lines when tested ex vivo in serum taken from these patients after antibody infusion, we postulate that the observed, prompt reduction of individual tumour cells in bone marrow was due to the cytotoxic action of the injected antibody. Although monitoring micrometastatic cells in bone marrow of patients with high tumour cell counts appears to be feasible, the immunocytochemical assay needs to be improved for patients with lower cell numbers before it can be applied as a surrogate test for adjuvant therapies.

Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody.

Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells, and did not use either L(ey) epitopes on target cells for cross-linkage of virus to the cell or the Fc part of the antibody as a ligand for any cellular receptor. For enhancement to occur, binding of anti-Le(y) antibody to virus was required to take place before virus binding to its specific receptor with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364 also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti-Le(y) ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection, and that this function was abrogated by the IgG1 Fc of the chimeric and the humanized antibodies. The observations indicate that the non-paratope domains of antiviral antibodies can influence their function as neutralizing or enhancing for infection.

Immune response to tumor antigens in a patient with colorectal cancer after immunization with anti-idiotype antibody.

An active vaccination protocol was performed on one patient with colon carcinoma as a pilot to a prospective randomized double-blind clinical trial with the vaccine SDZ SCV 106. This vaccine is an anti-idiotype goat antibody to the monoclonal antibody 17-1A, which is directed against the tumor antigen 17-1A. To study the effect of the therapy on the immune reactivity, several tests were performed to detect anti-tumor antibodies in the serum as well as in eluates of metastatic tissue. Furthermore metastases removed from the lung were examined by immunohistochemistry. The results suggest that the humoral and cellular immune reactivity against the tumor are enhanced.

Enzymatic synthesis and comparative biological evaluation of a phosphonate analogue of the lipid A precursor.

Phosphonate analogue 5 of the lipid A precursor 4 has been prepared from phosphonate 2 and nucleotide 3 with the help of lipid A synthase, isolated from the overproducing Escherichia coli mutant MC 1061 (delta 2512) or JB1104 (delta 2514). The biological properties of phosphonate 5 and phosphate 4 are quite similar to each other as compared in the limulus amoebocyte lysate assay, by the activation of the RAW264 murine macrophagelike cell line (determined by stimulation of ornithine decarboxylase), and by the pyrogenicity in rabbits. Hydrolytic removal of the 1-phosphate group of 4 is thus not a prerequisite for its biological activity.

Polyclonal anti-idiotypic antibodies mimicking the small cell lung carcinoma antigen cluster-5A interact with a panel of antibodies and induce specific immune response in animals.

Polyclonal anti-idiotypic antibodies (ab2) were generated by immunising goats with the murine IgG2a monoclonal antibody SWA20 which recognises the SCLC antigen cluster-5A, a tumour-associated sialoglycoprotein. Ab2 was shown to bind specifically to antibody SWA20, but not to isotype matched control antibodies. Pre-incubation with ab2 completely inhibited target cell binding of antibody SWA20 and of four other antibodies to cluster-5A antigen, while no effect was seen with antibodies to cluster-1 and cluster-w4 antigen. By these criteria the ab2 population consists of antibodies resembling in their reactivity pattern the cluster-5A antigen. Ab2 was used for immunisation of rabbits and two strains of mice; control animals received PBS or nonspecific goat IgG. Anti-anti-idiotype sera (ab3) were analysed in a series of radioimmunoassays for reactivity with goat IgG and reactivity with ab2. After blocking the nonspecific anti-goat response, ab3 could be shown to bind specifically to ab2 idiotype. As examined by an indirect cell ELISA with fixed cells, two out of six ab3 sera showed significantly higher binding ratios to antigen-positive SW2 cells as compared to antigen negative control cells. These findings indicate that the goat anti-SWA20 idiotype antibodies functionally represent the SCLC antigen cluster-5A and therefore may have potential for modulating the anti-tumour response through idiotypic network interactions.

Tumor cell lysis and tumor growth inhibition by the isotype variants of MAb BR55-2 directed against Y oligosaccharide.

IgG3 murine MAb BR55-2 is directed against adenocarcinoma associated Y oligosaccharide. Isotype IgG1, IgG2b and IgG2a switch variants of the BR55-2 antibody were compared in antibody dependent-cell mediated cytotoxicity (ADCC) and complement mediated cytotoxicity (CDC) assays in the murine system. IgG3, IgG2a and IgG2b isotypes mediated ADCC with murine macrophages. Murine splenocytes mediated low levels of ADCC, with IgG3 and IgG2a being always more effective than IgG1 and IgG2b isotypes. All four isotypes were ineffective in CDC with murine serum as a source of complement. In the in vivo experiments, growth of human tumors xenotransplanted into nude mice was best inhibited by both IgG3 and IgG2a isotypes while IgG1 protein was the least effective. Thus, IgG3 and IgG2a isotypes are the most efficient proteins for cancer immunotherapy since they mediate the highest tumoricidal activities in vitro and in vivo.

Biological activity in the human system of isotype variants of oligosaccharide-Y-specific murine monoclonal antibodies.

The capacity of isotype variants of BR55-2, an anti-tumor monoclonal antibody directed against Y oligosaccharide, to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in the human system was evaluated using freshly isolated peripheral blood mononuclear cells, lymphocytes, monocytes and complement. The ADCC activities of the BR55-2 IgG3 isotype and its switch variants (IgG1, IgG2b, and IgG2a) with human monocytes were high for all isotypes, whereas the activity of all isotypes was lower with freshly isolated lymphocytes, IgG1 being the least effective. The CDC on the other hand was strong with IgG3, IgG2b and IgG2a and negative with the IgG1 variant. The IgG3 and IgG2a isotypes were selected for further development. Their strong ADCC and CDC activity against mammary carcinoma, colon carcinoma and small-cell lung tumor cell lines was confirmed quantitatively using proteins highly purified from tissue-culture supernatants. Significant variations in ADCC and CDC competence was observed among human donors.

Oligosaccharide Y specific monoclonal antibody and its isotype switch variants.

Isotype switch variants of the IgG3 secreting hybridoma BR55-2 were developed with gamma 1, gamma 2b and gamma 2a heavy chains. The variants have the same affinity for antigen and expected affinities for the Fc receptor. The Y antigen defined by MAb BR55-2 has a restricted distribution to breast, colorectal, pancreatic, gastric and small cell lung carcinomas. The MAb BR55-2 also bound to prostatic and laryngeal carcinomas. The BR55-2 defined epitope is expressed by gastrointestinal cancer cell lines as a glycolipid, while the breast carcinoma cells express the same epitope on glycoproteins. These differences may influence tumor cell susceptibility to lysis by MAb BR55-2 and its isotype switch variants.

Role of the 1-amino group in aminocyclitol antibiotics: synthesis of 1-deaminogentamicin C2.

The synthesis of 1-deaminogentamicin C2 described here, uses 3,2',6',3"-tetrakis-N-tert-butoxycarbonylgentamicin C2 (2) as intermediate. N-Formylation of 2 followed by per-O-acetylation and dehydration furnished the isocyanide 5. Radical-induced deamination of the latter using tri-n-butylstannane and removal of the protecting groups afforded the target 1-deaminogentamicin C2 (7). Its in vitro antibacterial activity is less than that of the parent gentamicin C2. The behaviour of 7 towards aminoglycoside-inactivating enzymes was also examined; interestingly, it was found to be neither substrate nor inhibitor for such enzymes. These results strongly suggest that the substitution pattern of the 1-position determines the biological properties of the aminoglycoside antibiotics.

1-N-acylation of gentamicin C1a by a cyclic, chiral gamma-amino-alpha-hydroxy acid related to the (S)-4-amino-2-hydroxybutyric acid.

A semisynthetic aminoglycoside antibiotic 15, containing a cyclic gamma-amino-alpha-hydroxy acid, related to the 1-N-4-amino-2-hydroxybutyric acid (AHBA) side chain of butirosins and amikacin, has been prepared. Conveniently protected 3,2',6'-tris-N-tert-butoxycarbonylgentamicin C1a (12) was condensed with the phtalimido active ester 10 to give after catalytic reduction and deprotection, the hitherto unknown 1-N-substituted gentamicin C1a 15. The requisite side chain was synthesized from the readily available D-(-)-quinic acid. The antibacterial properties of 15 are given.