Use Of Human Antibodies Research Articles

Use of anti-gSG6-P1 IgG as a serological biomarker to assess temporal exposure to Anopheles' mosquito bites in Lower Moshi.

BackgroundMalaria prevalence in the highlands of Northern Tanzania is currently below 1% making this an elimination prone setting. As climate changes may facilitate increasing distribution of Anopheles mosquitoes in such settings, there is a need to monitor changes in risks of exposure to ensure that established control tools meet the required needs. This study explored the use of human antibodies against gambiae salivary gland protein 6 peptide 1 (gSG6-P1) as a biomarker of Anopheles exposure and assessed temporal exposure to mosquito bites in populations living in Lower Moshi, Northern Tanzania.MethodsThree cross-sectional surveys were conducted in 2019: during the dry season in March, at the end of the rainy season in June and during the dry season in September. Blood samples were collected from enrolled participants and analysed for the presence of anti-gSG6-P1 IgG. Mosquitoes were sampled from 10% of the participants’ households, quantified and identified to species level. Possible associations between gSG6-P1 seroprevalence and participants’ characteristics were determined.ResultsThe total number of Anopheles mosquitoes collected was highest during the rainy season (n = 1364) when compared to the two dry seasons (n = 360 and n = 1075, respectively). The gSG6-P1 seroprevalence increased from 18.8% during the dry season to 25.0% during the rainy season (χ2 = 2.66; p = 0.103) followed by a significant decline to 11.0% during the next dry season (χ2 = 12.56; p = 0.001). The largest number of mosquitoes were collected in one village (Oria), but the seroprevalence was significantly lower among the residents as compared to the rest of the villages (p = 0.039), explained by Oria having the highest number of participants owning and using bed nets. Both individual and household gSG6-P1 IgG levels had no correlation with numbers of Anopheles mosquitoes collected.ConclusionAnti-gSG6-P1 IgG is a potential tool in detecting and distinguishing temporal and spatial variations in exposure to Anopheles mosquito bites in settings of extremely low malaria transmission where entomological tools may be obsolete. However studies with larger sample size and extensive mosquito sampling are warranted to further explore the association between this serological marker and abundance of Anopheles mosquito.

Effects of a second-generation human anti-ErbB2 ImmunoRNase on trastuzumab-resistant tumors and cardiac cells

The inhibition of ErbB2 by the use of human antibodies can be a valuable strategy for the treatment of breast and gastric cancer. Trastuzumab, a humanized anti-ErbB2 antibody in clinical use, is effective but can engender resistance as well as cardiotoxicity. ImmunoRNases, made up of a human anti-ErbB2 scFv and human pancreatic ribonucleases (HP-RNases), have been engineered to overcome the limits of other immunotoxins, such as immunogenicity and nonspecific toxicity. Here, we report that a novel anti-ErbB2 immunoRNase, called Erb-HPDDADD-RNase, obtained by fusing Erbicin, a human ErbB2-directed scFv, with an HP-RNase variant that resists the cytosolic inhibitor protein, binds with high affinity to a panel of ErbB2-positive gastric tumor cells and inhibits their growth more than does the parental immunoRNase, which is not resistant to the inhibitor. Moreover, Erb-HP-DDADD-RNase is endowed with antiproliferative activity for trastuzumab-resistant cancer cells both in vitro and in vivo that is more potent than that of the parental immunoRNase. Importantly, Erb-HP-DDADD-RNase does not show cardiotoxic effects in vitro on human cardiomyocytes and does not impair cardiac function in a mouse model. Thus, Erb-HP-DDADD-RNase could fulfil the therapeutic need of cancer patients ineligible for trastuzumab treatment due to primary or acquired trastuzumab resistance or to cardiac dysfunction.

Effects of a human compact anti-ErbB2 antibody on prostate cancer

Prostate cancer is the most commonly diagnosed malignancy in men in developed countries. ErbB2, a tyrosine kinase receptor overexpressed in many human cancer types, contributes to prostate cancer progression by activating the androgen receptor in a steroid poor environment, thus promoting androgen-independent cell growth. The consequent development of hormone refractory tumors is a major obstacle in prostate cancer therapy. The inhibition of ErbB2 signal transduction pathways by the use of human antibodies could be a valuable alternative strategy for cancer therapy. We performed a comparative analysis in vitro and in vivo of the antitumor effects of three different antibodies targeting different epitopes of ErbB2: Herceptin (trastuzumab), 2C4 (pertuzumab) and Erb-hcAb (human anti-ErbB2-compact antibody), a novel fully human compact antibody produced in our laboratory. Herein, we demonstrate that the growth of both androgen-dependent and independent prostate cancer cells was efficiently inhibited by Erb-hcAb. The antitumor effects induced by Erb-hcAb on some cell lines were more potent than those observed for either Herceptin or 2C4. Thus, Erb-hcAb could be a promising candidate in the immunotherapy of prostate cancer for which no obvious treatment has been reported so far.

In-Vitro-Effects of TNF-A and IL-1-Antagonists on the Cytokine Network of Patients with Oligoarticular Idiopathic Arthritis (OJIA)

Background and aims: Pro-inflammatory cytokines are the main mediators of juvenile idiopathic arthritis. Modern therapeutic strategies (biologicals) are therefore geared to block the action of individual profinflammatory cytokines selectively. We investigated the effect of selective cytokine blocking on the equlilibrium of associated inflammatory mediators. Methods: Lipopolysaccharid stimulation was performed in vitro on peripheral mononuclear cells from 10 patients with oJIA and 15 healthy individuals for 24 hours. Etanercept, Anakinra and Adalimumab were used to inhibit cytokine effects. In the collected culture supernatants the profile of cytokine production was studied by multiplex cytokine bead immunoassay. Results: In healthy individuals selective cytokine inhibition prevented proinflammatory cytokine production collectively. In oJIA cell cultures with Adalimumab or Anakinra TNF-α or IL-1β concentrations were significantly decreased. In contrast this could not be shown for Etanercept (TNF-α). We found decreased amounts of proinflammatory cytokines IL-1β and IFN-Y. A decrease of IL-6 could be achieved only with Anakinra and Etanercept. Interestingly, after blockade of TNF-α or IL-1β we found increases in concentrations of proinflammatory IL-8 and anti-inflammatory IL-4. Conclusions: Blockade of cytokine effects of TNF-α or IL-1β may cause the decline of further proinflammatory cytokines in oJIA patients. This selective anti-inflammatory effect is likely supported by an increase of IL-4. The simultaneous presence of proinflammatory IL-8 seems to account for the maintenance of autoinflammation. This could be an attempt to explain the pathogenesis of therapy-resistant disease progression in anti-cytokine therapy. Hence, the use of humanized antibodies against IL-8 may play a role in future treatment of JIA.

The clinical utility of cytotoxic T lymphocyte antigen 4 abrogation by human antibodies

The recent cloning and identification of a variety of regulatory and counter-regulatory molecules on T cells and antigen presenting cells has led to the development of antibodies and other molecules that either stimulate or abrogate these immune functions. Patients with autoimmune disease, graft rejection and cancer might benefit from the ability to manipulate immune regulatory pathways. The first demonstration of clinical benefit by modulation of immune regulation in cancer involves the use of human antibodies against cytotoxic T lymphocyte antigen 4. Murine preclinical studies suggested that cytotoxic T lymphocyte antigen-4 abrogation would provide clinical benefit after an antitumor vaccination. Early trials of this antibody in patients with melanoma have shown antitumor activity with and without vaccines that is associated with a state of autoimmunity. Surprisingly, the reversal of the state of autoimmunity induced by cytotoxic T lymphocyte antigen 4 antibodies by the use of corticosteroids does not eliminate clinical benefit.

Potential use of humanized antibodies in the treatment of breast cancer

With the growing knowledge of key cellular pathways in tumor induction and evolution, targeted therapies make up an increasing proportion of new drugs entering clinical testing. In the treatment of breast cancer, humanized antibodies have become a major option. The humanized monoclonal antibody trastuzumab (Herceptin®; Genentech, Inc., CA, USA) for HER2-overexpressing, metastatic breast cancer, represents a successful agent associated with impressive survival benefits when combined with chemotherapy. Based on impressive results, trastuzumab will become a standard in the adjuvant treatment of HER2-overexpressing breast cancer. The role of trastuzumab in the neoadjuvant setting is promising, but must be further evaluated in large prospective, randomized trials. However, there is still a large proportion of patients overexpressing HER2 that do not respond to trastuzumab. Regarding this patient cohort, the optimal combination of trastuzumab with other agents needs further evaluation. In breast cancer lacking HER2 amplification, the role of the new antibody pertuzumab remains to be defined. The role of antibodies interfering with angiogenesis, tumor stroma or glycoproteins is of a preliminary nature and warrants further investigation. Here, an overview of humanized antibodies in human breast cancer is provided, with emphasis on the recent advances and future prospects in treating malignant breast cancer.

New immunosuppressants in clinical trial.

Chemical immunosuppression developed at the beginning of clinical transplantation remains the basis of immunosuppressive protocols used today. Clinical trials of new agents focus on the incremental improvement in one of four areas of immunotherapy: induction, basic maintenance, adjuvant and steroid therapy. Induction immunotherapy developments include the use of humanized antibodies, specific targets (basiliximab, daclizumab) and lymphocyte depletion (anti-thymocyte globulin, campath-1H). Promising results from pilot trials allow for subsequent low dose maintenance monotherapy. New inhibitors of calcineurin isoforms (ISATx247) may disassociate the anti-rejection effect from the toxicity of conventional calcineurin inhibitors. Revived investigation of older anti-proliferative or anti-metabolite drugs (rapamycin, mycophenolic acid, leflunomide) show promise in preventing B-cell responses and reducing chronic rejection but development of derivatives (everolimus, mycophenolate mofetil, ERL 080, MNA 279, MNA 715) may have a stronger commercial than experimental basis. Trials of steroid immunotherapy have focused on steroid elimination but trial of newer locally active steroids may be beneficial.

Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

Use of human antibodies to identify antigens in cultured human tumor cells: Detection of discrete antigen molecules using electroblotting and enzyme-linked antibody probes

To demonstrate antigens in cultured human tumor cells recognized by antibodies in human sera, extracts of cultured malignant melanoma cells were separated by SDS-PAGE, and electrophoretically transferred to nitrocellulose paper (NCP). The resulting electroblots were incubated with human serum, and bound immunoglobulin (Ig) was visualized with rabbit antibodies specific for human IgG, IgA or IgM, followed by peroxidase-conjugated goat anti-rabbit IgG. Antigen-antibody reactions in the nitrocellulose paper were also detected using 125I-labeled anti-rabbit IgG. As little as 125 pg of bound antibody per band were detectable. The numbers of proteins recognized by antibodies in human sera depended both on the quantity of protein transferred and the concentration of Ig applied to the NCP. Whole serum could not be used at dilutions ≤ 1:20 without an unacceptable increase in background staining. Binding of Ig to tumor cell proteins transferred to NCP depended on interactions with the Fab′, not the Fc region of the Ig molecule. To determine the efficiency of transfer as a function of both time and molecular weight, tumor cell proteins were intrinsically labeled with 75Se-labeled methionine and transferred for up to 4 h after fractionation in gels containing acrylamide concentrations of 5%, 7.5%, 10% or 12%. Proteins <150 kDa were transferred with particularly high efficiency in ≥2 h. Different antigens were recognized by the IgA, the IgG and the IgM molecules from the same sera. The methods outlined herein are proving to be useful in monitoring the purification of specific antigens from whole tumor cell extracts.

Simplified immunoradiometric assay for factor VIII coagulant antigen.

A simplified, non-competitive, solid phase immunoradiometric assay has been developed for the quantitation of factor VIII coagulant antigen (VIII:CAg)--the antigenic counterpart of FVIII coagulant activity (VIII:C). Both homologous and heterologous antibodies to human factor VIII (FVIII) were used in this assay. Initially, FVIII in a test sample was attached to immobilized, human IgG obtained from a polytransfused haemophilia A patient with a high titre antibody to VIII:C. The bound FVIII was then detected using rabbit 125I-IgG specific for human FVIII. The concentration of VIII:CAg correlated well with VIII:C levels in the plasma from normal donors (r = 0.84, n - 15). Homozygote von Willebrand's disease patients had undetectable levels of VIII:CAg in their plasma. Patients with severe haemophilia A (VIII:C less than 0.01 u/ml) could be divided into groups on the basis of the VIII:CAg levels, i.e. those having undetectable VIII:CAg and other with measurable VIII:CAg. VIII:CAg detected in normal serum was less than 0.002 u/ml. In this assay the use of human antibody to FVIII is considerably decreased compared to other methods for VIII:CAg, and the time-consuming steps to immunopurify human anti-FVIII antibody are eliminated.

Antinuclear antibodies--attempts at ultrastructural localization.

Considerable interest has evolved over the last 10 years concerning the raison d'etre as well as the pathogenic significance of antinuclear antibodies among human disease states. The elution from isolated lupus glomeruli of antibodies showing distinct anti-DNA or anti-nucleoprotein reactivity (1-3) has produced strong evidence that these γ-globulins may participate as part of antigen-antibody complexes localizing within glomerular lesions. Recently, by use of human antibodies to DNA and immunofluorescent procedures, it has been shown that DNA itself is present within some glomerular lesions as the possible antigenic contribution to pathogenic complexes (4). Moreover, eluted γ-globulin from lupus glomeruli was shown by these same workers to contain marked concentrations of antibody activity for various nuclear antigens.The present study was primarily directed at an electron microscopic localization within the nucleus of high titered antinuclear antibodies using sera from patients with active disseminated l...