Use of antibodies to the lipopolysaccharide core region in the treatment of gram-negative sepsis and shock

In this paper we review evidence in favor of a protective role for antibodies directed at the lipopolysaccharide (LPS) core region of Gram-negative bacilli. It will be shown that, given the right animal model, polyclonal antisera raised against O-antigen lacking, rough mutant strains, can be shown to protect animals against lethal sepsis due to a variety a bacterial species. Evidence for a protective role obtained from clinical studies will also be discussed. It will be stressed how difficult it is to prove that the antibodies directed against the LPS-core, and not other proteins present in the polyclonal anti-rough strain antisera, were responsible for the observed protective effects. The solution to some of these problems is the production of monoclonal antibodies (Mabs). We will discuss the findings that purified anti-core Mabs may give false-positive outcomes of protection studies, due to the induction of tolerance by small amounts of contaminating endotoxin, present in the antibody preparation. The need to administer the Mab therapeutically instead of prophylactically to the test animals, will be stressed. It will be shown how we succeeded in determining the epitope-specificities of several anti-core antibodies. It will be shown also that use of Elisa alone for characterization of anti-core Mabs may lead to false conclusions concerning epitope specificities. We will prove that synthetic KDO-(=ketodeoxycctanate) and lipid A-containing antigens are indispensable tools for thorough characterization of Mabs. Using this methodology we were able to discriminate between specific antigen-antibody interactions, and the ability of some Mabs to bind “non-specifically” to hydrophobic surfaces. The relationship between epitope specificity and protective effects of anti-core Mabs will be described. Finally, we will demonstrate that a KDO-specific Mabs is capable of protecting mice against lethal sepsis; in other words, it will be shown conclusively that Mabs to defined epitopes in LPS core region are cross-protective.     

Targetted localisation and imaging of a murine lymphoma using 131I-labelled monoclonal antibody

In vivo tumor targetting with radiolabelled monoclonal antibodies is a promising approach for the diagnosis and therapy of tumors. A specific monoclonal antibody (mAb), DLAB was generated to the Dalton's lymphoma associated antigen (DLAA) from Haemophilus paragallinarum-induced spontaneous fusion. In order to study the tumor localisation and biodistribution properties of the monoclonal antibody, scintigraphic studies were performed using the radiolabelled DLAB. 131-labelled DLAB was administered intravenously into Swiss mice bearing Dalton's lymphoma and external scintiscanning was performed at different time intervals. Clear tumor images were obtained which revealed selective and specific uptake of radiolabel and the results were compared with biodistribution data. The radioiodinated monoclonal antibody showed fast tumor uptake which increased significantly to 14.6% injected dose (ID)/g at 12 hr post-injection. Enhanced blood clearance of radioactivity resulted in higher tumor/blood ratio of 5.96 at 48 hr. 131I-labelled DLAB resulted in selective and enhanced uptake of the radioactivity by the tumor compared to the non-specific antibody and the results suggest the potential use of spontaneous fusion for producing specific monoclonal antibodies for tumor detection and therapy.     

Monoclonal antibodies in diagnostics of high-grade gliomas

The review considers the modern methods of radioimmune diagnostics of high-grade gliomas using monoclonal antibodies and also other approaches of multi-component radioimmunolocalization with pretargeted bispecific antibodies. High-grade tumor-related proteins have been analyzed as potential targets for radiolabeled antibodies. Recent experimental and clinical data on immunolocalization of brain tumors and the most promising immunochemical approaches for diagnostic and targeted therapy of gliomas with tumor-specific antibodies are discussed.     

Clinical Value of Teichoic Acid Antibody Titers in the Diagnosis and Management of the Staphylococcemias

Differentiation of endocarditic from nonendocarditic Staphylococcus aureus (SA) septicemia is prognostically and therapeutically important. A study of 68 cases of either SA or streptococcal sepsis, including 50 cases of SA sepsis of both cardiac and noncardiac origin, was done to determine the presence and titer of serum teichoic acid antibodies (TAA''s) by double immunodiffusion. Thirty-seven uninfected controls were also examined. There was no statistical difference in either incidence or peak TAA titers in endocardial versus deepseated, extracardiac SA sepsis. However, in both of these groups, incidence and peak titers were significantly higher than in intravascular catheter-related SA sepsis, streptococcal endocarditis and controls (P<0.05). Peak TAA titers in SA sepsis develop on admission or shortly thereafter (6 to 11 days) and permit early decisions on degree of tissue infection, likelihood of metastatic seeding and necessity for higher-dose, longer-term antibiotic therapy.Cases of catheter-related SA sepsis with no clinical evidence of metastatic SA seeding and with negative or low-titered (1:1) TAA''s were classified as superficial sepsis. Treatment consisted of short-term, low-dose antistaphylococcal regimens and catheter removal. In posttherapy follow-up after 6 to 12 weeks, all of the patients were cured and no signs of endocarditis or deepseated SA infection developed.     

Antigenic determinants of lipid A analyzed with synthetic models and monoclonal antibodies

Three monoclonal antibodies directed against the hydrophobic region (lipid A backbone) of Bordetella pertussis endotoxin were used to analyze cross reactivities of lipid A and synthetic glycolipids structurally related to it. Reactivity of mAb with synthetic compounds was measured by a radioimmunoassay inhibition test. Our data indicate that 3-acyloxyacyl groups do not play a significant role, and suggest that a phosphate and a hydroxyl group, at two carbon distance from each other, are part of a single antigenic determinant recognized by the monoclonal antibodies.     

抗CD20单克隆抗体治疗B 细胞淋巴瘤的效应机制

B细胞淋巴瘤是一种主要的非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL),95%以上的B细胞淋巴瘤均表达B细胞分化抗原CD20。尽管目前CD20在B细胞分化发育过程中的具体功能尚未阐明,但其特有的表达方式和在细胞膜上的分布特点决定了其成为B细胞淋巴瘤靶向治疗的主要靶点。近十年来,随着抗CD20单克隆抗体的不断发展和进步,联合传统的CHOP化疗方案,在NHL的治疗中显示出良好的效果。虽然,近年来抗CD20单抗逐渐被用于B细胞相关的自身免疫病的治疗,但相关作用机制尚不明确。在针对NHL的临床治疗中,抗CD20单抗的疗效被认为依赖于效应器机制,主要有ADCC、CDC和细胞凋亡。虽然抗CD20在淋巴瘤的治疗中具有显著的免疫疗效,但部分瘤荷较大的病人出现了耐药和复发,循环中大量B细胞由于单抗的结合而被机体的单核巨噬细胞吞噬或NK细胞杀伤,机体出现成熟B细胞空缺期,同时单核巨噬细胞、NK细胞和补体大量消耗,造成机体免疫效应功能饱和和效应器耗竭。本文就抗CD20单克隆抗体在治疗淋巴瘤中的具体作用机制及可能造成的机体效应器耗竭问题做一简要的概述。     

Antibiotic-induced release of inflammatory mediators from bacteria in experimentalKlebsiella pneumoniae-induced sepsis

In a fibrin-clot model of sepsis, developed in mice, treatment with the antibiotics ceftazidime (Cfz) and ofloxacin (Ofl) caused significant (p < 0.01) release of endotoxin and TNF-alpha after 4.5 h when compared with control (untreated) and amikacin (Ami) treated group. Except for control group, the level of bacteremia declined in all three antibiotic-treated groups. The results suggest that antibiotic therapy, irrespective of the agent used, results in an increase in endotoxin levels in vivo. The amount of endotoxin liberated by Ami was much smaller than with Cfz and Ofl therapy, which makes it an appropriate agent for the treatment of sepsis. An increase in the level of TNF-alpha along with endotoxin is suggestive of increased inflammatory response.     

Tumor-directed targeting of liposomes

Tumor-specific targeting is a critical goal in the research area of liposomal drug delivery. Identification of the specific interactions between ligands and target tumor cells is a principle prerequisite in achieving this goal. Generally, tumor cells aberrantly express tumor-associated antigens that can be utilized as appropriate target molecules. Monoclonal antibodies against tumor-associated antigens have been successfully adopted for targeting to various types of cancer cells. The incorporation of humanized monoclonal antibodies or single chain human antibodies, instead of rodent antibodies into immunoliposomes has resulted in better clinical applicability. Tumor-specific ligands other than monoclonal antibodies have also been investigated as in vivo tumor-directing molecules. However, the number of pre-clinical studies of anticancer treatments using tumor-specific liposomal drugs reporting successful targeting and enhanced therapeutic efficacy has been limited. Further refinement of tumor-specific interactions and liposomal formulations will be necessary for the application of the tumor-specific liposomal drug strategy for anticancer chemotherapy or gene therapy.     

Monoclonal antibodies in solid tumours: approaches to therapy with emphasis on gynaecological cancer

Monoclonal antibodies have progressed from the laboratory to the clinic. Although recognised in diagnosis there are still problems as far as their therapeutic use is concerned. This review looks at the history, principles of active specific immunotherapy, clinical experience with monoclonal antibodies in therapy of solid tumours, in particular the development of new bispecific monoclonal antibodies, and trials in ovarian, breast and colorectal cancer. Immunoconjugates, linked with radionuclides and cytotoxic drugs, indicate future developments. Conditions for successful therapy, especially with adjuvants in patients with small tumour residues, are also described.     

miR-29b-3p protects cardiomyocytes against endotoxin-induced apoptosis and inflammatory response through targeting FOXO3A

Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.     

Biopanning of endotoxin-specific phage displayed peptides

Systemic bacterial infections frequently lead to a plethora of symptoms termed "endotoxic shock" or "sepsis." Characterized by hypotension, coagulation abnormalities, and multiple organ failure, treatment of sepsis still remains mostly supportive. Of the various experimental therapeutic interventional strategies, neutralization of endotoxin by peptides or proteins is becoming popular recently. Hence, design of endotoxin binding peptides is gaining currency as their structural complexity and mode of recognition of endotoxin precludes mounting of resistance against them by the susceptible bacteria by genetic recombination, mutation, etc. Earlier work from our laboratory had shown that the amphiphilic cationic peptides are good ligands for endotoxin binding. In this study, we report the results of studies with the 12 selected lipid A binding phage displayed peptides by biopanning of a repertoire of a random pentadecapeptide library displayed on the filamentous M-13 phage. A comparison of the sequences revealed no consensus sequence between the 12 selected peptides suggesting that the lipid A binding motif is not sequence specific which is in accord with the sequence variation seen with the naturally occurring anti-microbial and/or endotoxin binding peptides. Thus, the flexibility of the peptides coupled with their plasticity in recognizing the lipid A moiety, explains their tight binding to endotoxin. At a structural level, asymmetric distribution of the charged polar residues on one face of the helix and non-polar residues on the opposite face appears to correlate with their activity.     

Micrometastatic bone marrow involvement: detection and prognostic significance

The present review focuses on the methodology and clinical significance of new diagnostic approaches to identify individual cancer cells present in bone marrow, both as a frequent site of metastasis formation and an indicator organ for hematogenous tumor cell dissemination. The steadily increasing number of studies on this issues is characterized by considerable methodological variations of important variables, such as the size of the study population, and the reliability of monoclonal antibodies used for tumor cell detection. Emerging data indicate that this disturbing heterogeneity might be overcome by the use of reliable and specific anti-cytokeratin antibodies (for example, A45-B/B3) as, for the time, standard markers for the detection of micrometastatic tumor cells in bone marrow. Prospective clinical studies have shown that immunoassays based on anti-CK antibodies identify patients’ subgroups with a poor clinical prognosis with regard to early metastasis manifestation and reduced overall survival in various epithelial tumor entities, including breast, colon, rectum, stomach, esophagous, prostate, renal, blasdder, and nonsmall cell lung cancer. The immunocytochemical assays may be therefore used to improve tumor staging with potential consequences for adjuvant therapy, because disseminated cells appeared to be dormant, non-cycling (for example Ki-67 antigen-negative) cells, suggesting a resistance to cell-cycle dependent therapy, such as chemotherapy. Therefore, cell-cycle independent antibody-based immunotherapy might be an interesting option to complement chemotherapy. Another promising clinical application is monitoring the response of micrometastatic cells to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical followup. The outlined current strategies for detection and characterization of cancer micrometastasis might help to design and control new therapeutic strategies for secondary prevention of metastatic relapse in patients with operable primary carcinomas.     

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

Background

Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein.

Results

In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody.

Conclusion

The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.     

Systemic vaccination with anti‐oligomeric monoclonal antibodies improves cognitive function by reducing Aβ deposition and tau pathology in 3xTg‐AD mice

Alzheimer's disease (AD) is a devastating disorder that is clinically characterized by a comprehensive cognitive decline. Accumulation of the amyloid‐beta (Aβ) peptide plays a pivotal role in the pathogenesis of AD. In AD, the conversion of Aβ from a physiological soluble monomeric form into insoluble fibrillar conformation is an important event. The most toxic form of Aβ is oligomers, which is the intermediate step during the conversion of monomeric form to fibrillar form. There are at least two types of oligomers: oligomers that are immunologically related to fibrils and those that are not. In transgenic AD animal models, both active and passive anti‐Aβ immunotherapies improve cognitive function and clear the parenchymal accumulation of amyloid plaques in the brain. In this report we studied effect of immunotherapy of two sequence‐independent non‐fibrillar oligomer specific monoclonal antibodies on the cognitive function, amyloid load and tau pathology in 3xTg‐AD mice. Anti‐oligomeric monoclonal antibodies significantly reduce the amyloid load and improve the cognition. The clearance of amyloid load was significantly correlated with reduced tau hyperphosphorylation and improvement in cognition. These results demonstrate that systemic immunotherapy using oligomer‐specific monoclonal antibodies effectively attenuates behavioral and pathological impairments in 3xTg‐AD mice. These findings demonstrate the potential of using oligomer specific monoclonal antibodies as a therapeutic approach to prevent and treat Alzheimer's disease.     

Analytical comparability study of recombinant monoclonal antibody therapeutics

Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.     

临床诊断与治疗的新工具——可变淋巴细胞受体

单克隆抗体(Monoclonal antibody, mAb)在癌症以及自身免疫等疾病的诊断与治疗中得到广泛应用, 并且取得了重大进展。当今应用于临床的单克隆抗体是在免疫球蛋白的基础上进行改造研发而得。然而近期发现的无颌类脊椎动物的特异性抗原受体-可变淋巴细胞受体(Variable lymphocyte receptor, VLR), 为抗体类试剂或药物的研发提供了新的视角。与免疫球蛋白(Immunoglobulins, Ig)相比, VLR与抗原结合的特异性、亲和力及稳定性都优于Ig类抗体, 并且抗原特异性单克隆VLR的制备技术日趋成熟。因此, VLR在临床诊断和治疗中具有更高的应用价值, 并可能成为新一代的抗体药物。文章就VLR的基本特征、制备方法及其应用前景进行综述, 为实现VLR在临床诊断与治疗等领域中的应用提供有益参考。     

Interaction of IgG3 anti-streptococcal group A carbohydrate (GAC) antibody with streptococcal group A vaccine: enhancing and inhibiting effects of anti-GAC, anti-isotypic, and anti-idiotypic antibodies

Enhancement of binding of one monoclonal antibody to an antigen in the presence of a second monoclonal antibody (specific for an independent epitope on the same antigen) has been observed for several antigen-antibody systems involving primarily protein, or glycoprotein, antigens. We have analyzed the interaction between radiolabeled IgG3 kappa anti-streptococcal group A carbohydrate (GAC) antibody (125I-HGAC 39) and streptococcal group A vaccine (GAV; traditionally used to elicit anti-GAC antibody) in the absence and presence of unlabeled anti-GAC antibodies, anti-isotypic antibodies, or anti-idiotypic antibodies, respectively. A variety of significant enhancing or inhibiting effects on the binding of 125I-HGAC 39 to solid-phase GAV (GAVsp) were noted. First, high concentrations of IgG3 anti-GAC antibodies specifically inhibit binding of 125I-HGAC 39 to GAVsp, but the presence of lower concentrations of IgG3 anti-GAC antibodies is associated with markedly increased (up to 300 to 400%) binding of 125I-HGAC 39 to GAVsp. In contrast, with the concentrations used, IgM anti-GAC antibodies only inhibit binding of 125I-HGAC 39 to GAVsp. A monoclonal anti-gamma 3 antibody (2E.6) also enhances binding (up to 700%) of 125I-HGAC 39 to GAVsp, whereas another high-affinity anti-isotypic antibody, anti-C kappa (187.1), only inhibits binding of 125I-HGAC 39 to GAVsp. In a similar manner, an antiidiotypic antibody (anti-IdX) specific for a framework idiotope located near the C kappa domain inhibits the interaction between 125I-HGAC 39 and GAVsp. Evidence is presented to suggest that neither anti-C kappa nor anti-IdX blocks the HGAC 39 paratope, and therefore, the inhibition of binding mediated by these antibodies must be on some other basis. An alternative explanation for this effect, on the basis of the impairment of functional bivalency of 125I-HGAC 39, is discussed. Finally, anti-idiotypic antibodies (anti-IdI-3a and anti-IdI-1) that bind closer to the antigen-binding site of HGAC 39 inhibit binding of 125I-HGAC 39 to GAVsp in a manner that is most readily interpreted as competition for the GAC-binding site (or nearby sites) on the HGAC 39 variable domain. These effects are shown to require specific immunologic recognition of either GAVsp or 125I-HGAC 39.     

Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing

Immune sera from convalescent patients have been shown to be effective in the treatment of patients infected with Severe Acute Respiratory Syndrome Virus (SARS-CoV) making passive immune therapy with human monoclonal antibodies an attractive treatment strategy for SARS. Previously, using Xenomouse (Amgen British Columbia Inc), we produced a panel of neutralizing Human monoclonal antibodies (HmAbs) that could specifically bind to the ectodomain of the SARS-CoV spike (S) glycoprotein. Some of the HmAbs were S1 domain specific, while some were not. In this study, we describe non-S1 binding neutralizing HmAbs that can specifically bind to the conserved S2 domain of the S protein. However, unlike the S1 specific HmAbs, the S2 specific HmAbs can neutralize pseudotyped viruses expressing different S proteins containing receptor binding domain sequences of various clinical isolates. These data indicate that HmAbs which bind to conserved regions of the S protein are more suitable for conferring protection against a wide range of SARS-CoV variants and have implications for generating therapeutic antibodies or subunit vaccines against other enveloped viruses.     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

LPS and specific T cell responses: interleukin 1 (IL 1)-independent amplification of antigen-specific T helper (TH) cell proliferation

Lipopolysaccharide (LPS) fraction of endotoxin induces a significant potentiation of the antigen-specific proliferative response of T helper (TH) cell lines. This effect was obtained with LPS from different bacterial sources and reproduced with the lipid A moiety of endotoxin. Purified adherent spleen cells used as antigen-presenting cells (APC) support this LPS-enhanced TH cell proliferation. In addition, the effect of endotoxin on specific TH cell responses was found to be absolutely dependent on the interaction between TH lymphocytes and APC through antigen-specific recognition. Thus, it was not observed in the absence of specific antigen or when monoclonal antibodies against class II MHC products or against L3T4 antigens were used to inhibit the T cell-APC interaction. Similarly, it was found that APC from the B6.CH-2bm12 mutant do not support the LPS-mediated enhancing effect. Furthermore, interleukin 1 (IL 1) appears not to be involved in LPS-mediated enhancement, and this effect is not reproduced by muramyl dipeptide (MDP)-mediated activation of APC.